Microfluidic apparatus and methods of use thereof

ABSTRACT

Apparatuses and methods are described herein for processing polynucleotides in a sealed path environment. The apparatuses include optical sensors to monitor operations and to track material usage for good manufacturing practice.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication No. 62/885,159, filed on Aug. 9, 2019, and titled“MICROFLUIDIC APPARATUS AND METHODS OF USE THEREOF,” as well as U.S.Provisional Patent Application No. 62/885,170, filed Aug. 9, 2019, andtitled “METHODS AND APPARATUSES FOR MANUFACTURING THERAPEUTICCOMPOSITIONS,” and U.S. Provisional Patent Application No. 62/914,374,filed on Oct. 11, 2019, titled “METHODS AND APPARATUSES FORMANUFACTURING FOR REMOVING MATERIAL FROM A THERAPEUTIC COMPOSITION,”each of which is herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

FIELD

The apparatuses and methods described herein may be used for themanufacture and formulation of biomolecule-containing (includingtherapeutic mRNA) products, particularly therapeutics for individualizedcare. In particular, described herein are closed path methods andapparatuses for processing therapeutic polynucleotides, including at apoint of care.

BACKGROUND

Currently available technologies for manufacturing and formulatingpolynucleotide therapeutics, particularly mRNA therapeutics, oftenexpose the products to contamination and degradation. Currentlyavailable centralized production can be too costly, too slow, andsusceptible to contamination for use in therapeutic formulationspossibly including multiple polynucleotide species. Development ofscalable polynucleotide manufacturing, production of single patientdosages, elimination of touchpoints to limit contamination, input andprocess tracking for meeting clinical manufacturing requirements, anduse in Point-of-Care operations can advance the use of these promisingtherapeutic modalities. Microfluidic instrumentation and processes canprovide major advantages against these goals.

SUMMARY OF THE DISCLOSURE

Described herein are microfluidic apparatuses for manufacturingtherapeutics, including in particular mRNA therapeutics. For example,described herein are systems that may form therapeutic materials(including both drug substance and drug product) within a dedicated,single-use, disposable microfluidic path device (e.g., microfluidic pathplate device, chip, biochip, plate, etc.). Microfluidic path devices andcontrol systems for operating microfluidic path devices are described.

For example described herein are control systems (“apparatuses”) foroperating a microfluidic path device. These apparatuses may be referredto herein as microfluidic apparatuses, microfluidic control apparatuses,microfluidic control systems, or microfluidic systems.

A microfluidic apparatus may include: a seating mount (e.g., seat) for amicrofluidic path device; a plurality of fluid vials, wherein each fluidvial either comprises a fluidic line or is configured to couple with afluidic line, to form a closed fluid path; and a controller configuredto drive fluidic movement in the microfluidic path device when themicrofluidic path device is seated in the seating mount. In any of thesemicrofluidic apparatuses, each fluidic line may be configured to besealed against the microfluidic path device seated in the seating mountto form a closed fluid path. The microfluidic apparatuses describedherein may advantageously include any (or any combination of) thefeatures described herein.

For example, a microfluidic apparatus may include: a seating mount for amicrofluidic path device; a reagent storage frame comprising a pluralityof holders each configured to hold a fluid vial, wherein each fluid vialeither comprises a fluidic line or is configured to couple with afluidic line, further wherein each fluidic line is configured to bebiased against the microfluidic path device seated in the seating mountwith a bias force; and a controller configured to drive fluidic movementin the microfluidic path device when the microfluidic path device isseated in the seating mount.

A microfluidic apparatus may include: a seating mount for a microfluidicpath device; a fluid interface assembly comprising a plurality offluidic lines, wherein each fluidic line is configured to be separatelybiased against the microfluidic path device seated in the seating mountwith a bias force; a reagent storage frame comprising a plurality offluid sample holders each configured to hold a fluid vial and eachconfigured to couple to the fluid interface assembly through one of thefluidic lines of the fluid interface assembly; and a controllerconfigured to drive fluidic movement in the microfluidic path devicewhen the microfluidic path device is seated in the seating mount.

A microfluidic apparatus may include a seating mount for a microfluidicpath device; a plurality of pressure lines; a reagent storage framecomprising a plurality of holders each configured to hold a fluid vial,wherein each fluid vial either comprises a fluidic line or is configuredto couple with a fluidic line, further wherein each fluidic line andeach pressure line is configured to be biased against the microfluidicpath device seated in the seating mount with a bias force; and acontroller configured to control the application of pressure through thepressure lines to drive fluidic movement in the microfluidic path devicewhen the microfluidic path device is seated in the seating mount.

A microfluidic apparatus may include a seating mount for a microfluidicpath device; a fluid interface assembly comprising a plurality offluidic lines and pressure lines, wherein each fluidic line and eachpressure line is configured to be biased against the microfluidic pathdevice seated in the seating mount with a bias force; a reagent storageframe comprising a plurality of fluid sample holders each configured tohold a fluid vial and each configured to couple to the fluid interfaceassembly through one of the fluidic lines of the fluid interfaceassembly; and a controller configured to control the application ofpressure through the pressure lines to drive fluidic movement in themicrofluidic path device when the microfluidic path device is seated inthe seating mount.

A microfluidic apparatus may include a seating mount for a microfluidicpath device; a plurality of pressure lines; a plurality of fluid vials,wherein each fluid vial either comprises a fluidic line or is configuredto couple with a fluidic line, further wherein each fluidic line andeach pressure line is configured to be seal against the microfluidicpath device seated in the seating mount to form a closed fluid path; anda controller configured to control the application of pressure throughthe pressure lines to drive fluidic movement in the microfluidic pathdevice when the microfluidic path device is seated in the seating mount.

A microfluidic apparatus may include a seating mount for a microfluidicpath device; a plurality of fluid vials, wherein each fluid vial eithercomprises a fluidic line or is configured to couple with a fluidic line,further wherein each fluidic line is configured to be seal against themicrofluidic path device seated in the seating mount to form a closedfluid path: at least one optical sensor configured to monitor fluidwithin the microfluidic path device seated in the seating mount; and acontroller configured to drive fluidic movement in the microfluidic pathdevice when the microfluidic path device is seated in the seating mountand to record and/or transmit optical data showing fluid within themicrofluidic path during operation of the apparatus.

A microfluidic apparatus may include: a seating mount for a microfluidicpath device; a fluid interface assembly comprising a plurality offluidic lines and pressure lines, wherein each fluidic line and eachpressure line is configured to be biased (e.g., spring-loaded) againstthe microfluidic path device seated in the seating mount; a reagentstorage frame comprising a plurality of fluid sample holders eachconfigured to hold a fluid vial and each configured to couple to thefluid interface assembly through one of the fluidic lines of the fluidinterface assembly; and a controller configured to control theapplication of pressure through the pressure lines to drive fluidicmovement in the microfluidic path device when the microfluidic pathdevice is seated in the seating mount.

In some variations a microfluidic apparatus for processing therapeuticpolynucleotides at a point of care and configured to operate as a closedpath, may include: a seating mount for a microfluidic path device; afluid interface assembly comprising a plurality of fluidic lines andpressure lines, wherein each fluidic line and each pressure line isconfigured to independently be driven against the microfluidic pathdevice seated in the seating mount to make a sealing connection thereto;a reagent storage frame comprising a plurality of fluid sample holderseach configured to hold a fluid and each configured to couple to thefluid interface assembly through one of the fluidic lines of the fluidinterface assembly; a controller configured to control the applicationof pressure through the pressure lines to drive fluidic movement in themicrofluidic path device when the microfluidic path device is seated inthe seating mount; wherein the fluid interface assembly comprises aplurality of spring biases configured to independently urge each fluidicline against the microfluidic path device seated in the seating mount tomake a sealing connection thereto.

For example, an apparatus. (e.g., a microfluidic apparatus for forming atherapeutic polynucleotide) may include: a seating mount for removablyholding a microfluidic path device; a plurality of pressure lines; aplurality of fluid vials that are each pressurized by one or morepressure lines from the plurality of pressure lines, further whereineach fluid vial either comprises a fluidic line or is configured tocouple with the fluidic line, wherein each fluidic line and at least asubset of the pressure lines are configured to be independently biasedagainst the microfluidic path device seated in the seating mount to forma sealed closed fluid path; and a controller configured to control theapplication of pressure through the pressure lines to drive fluidicmovement in the microfluidic path device when the microfluidic pathdevice is seated in the seating mount and to apply pressure to one ormore of the pressure lines to open or close valves in the microfluidicpath device during operation.

As mentioned, in general, the controller may be configured to controlthe apparatus to perform an in vitro transcription (IVT) reaction in themicrofluidic path device.

For example, an apparatus (e.g., a microfluidic apparatus for forming atherapeutic polynucleotide, such as a therapeutic mRNA) may include: aseating mount for a microfluidic path device; a plurality of pressurelines; a fluid interface assembly comprising a plurality of fluidiclines; a plurality of fluid vials configured to be pressurized; areagent storage frame comprising a plurality of holders each configuredto hold a fluid vial of the plurality of fluid vials, wherein each fluidvial either comprises a fluidic line of the plurality of fluidic lines,or is configured to couple with a fluidic line of the plurality offluidic lines, further wherein each fluidic line and at least some ofthe pressure lines are configured to be separately biased against themicrofluidic path device seated in the seating mount with a bias force;and a controller configured to control the application of pressurethrough the pressure lines to drive fluidic movement in the microfluidicpath device when the microfluidic path device is seated in the seatingmount.

In some variations, the microfluidic apparatus (e.g., microfluidicapparatus for forming a therapeutic polynucleotide, such as atherapeutic mRNA) may include: a seating mount for removably holding amicrofluidic path device; a plurality of pressure lines wherein at leasta subset of the pressure lines are configured to be independently biasedagainst a pressure input on the microfluidic path device seated in theseating mount; a plurality of fluid vials configured to be pressurized,further wherein each fluid vial either comprises a fluidic outputconfigured to seal against an input on the microfluidic path device oris configured to couple with a fluidic line that is configured to beindependently biased against the microfluidic path device to form asealed closed fluid path; a first optical detector configured to monitorfluid within the fluid vials; a second optical detector configured tomonitor fluid within the microfluidic path device seated in the seatingmount; a controller configured to receive input from the first opticaldetector and the second optical detector and to control the applicationof pressure through the pressure lines to apply pressure from theplurality of pressure lines to open and/or close valves and to drivefluidic movement in the microfluidic path device based at least in parton the received input.

A microfluidic apparatus for processing therapeutic polynucleotides at apoint of care and configured to operate as a closed path may include: aseating mount for a microfluidic path device; a fluid interface assemblycomprising a plurality of fluidic lines and pressure lines, wherein eachfluidic line and each pressure line is configured to be independentlydriven against the microfluidic path device seated in the seating mountto make a sealing connection thereto; a reagent storage frame comprisinga plurality of pressurized fluid sample holders each configured to holda fluid vial and each configured to couple to the fluid interfaceassembly through one of the fluidic lines of the fluid interfaceassembly; a plurality of optical sensors arranged around the seatingmount and reagent storage frame to monitor fluid levels within the fluidvials held by the reagent storage frame and fluidic movement in themicrofluidic path device when the microfluidic path device is seated inthe seating mount; and a controller in communication with the opticalsensors and configured to control the application of pressure throughthe pressure lines to drive fluidic movement in the microfluidic pathdevice when the microfluidic path device is seated in the seating mount;wherein the fluid interface assembly comprises a plurality of colletsconfigured to independently urge each fluidic line against themicrofluidic path device seated in the seating mount to make a sealingconnection thereto, further wherein each of the seating mount, fluidinterface assembly and fluid sample holders are configured to beremovable for sterilization.

In some variations, these microfluidic apparatuses may be microfluidicapparatuses for forming a therapeutic polynucleotide (e.g., an mRNAtherapeutic). The apparatus may include: a seating mount for removablyholding a microfluidic path plate device, a plurality of pressure lines;a plurality of fluid vials, wherein each fluid vial either comprises afluidic line or is configured to couple with the fluidic line, whereineach fluidic line and at least a subset of the pressure lines areconfigured to be biased against the microfluidic path plate device heldin the seating mount to form a closed fluid path, and a controllerconfigured to control the application of pressure through the pressurelines to drive fluidic movement in the microfluidic path plate devicewhen the microfluidic path plate device is held in the seating mount,wherein the controller is configured to direct the synthesis of asynthetic template, direct an in vitro transcription (IVT) reactionusing the template to form a therapeutic polynucleotide, and directpurification of the therapeutic polynucleotide in one or moremicrofluidic path plate devices held in the seating mount.

A microfluidic apparatus (e.g., a microfluidic apparatus for forming atherapeutic polynucleotide, such as a therapeutic mRNA) may include: aseating mount for removably holding a microfluidic path plate device; aplurality of pressure lines; a plurality of fluid vials, wherein eachfluid vial either comprises a fluidic line or is configured to couplewith the fluidic line, wherein each fluidic line and at least a subsetof the pressure lines are configured to be biased against themicrofluidic path plate device held in the seating mount to form aclosed fluid path; and a controller configured to control theapplication of pressure through the pressure lines to drive fluidicmovement in the microfluidic path plate device when the microfluidicpath plate device is held in the seating mount, wherein the controlleris configured to determine the contents of the fluid vials, transfersub-microliter amounts of material from the fluid vials to one or morereactors in the microfluidic path plate device held in the seatingmount, direct the synthesis of a synthetic template, direct an in vitrotranscription (IVT) reaction using the template to form a therapeuticpolynucleotide, and direct purification of the therapeuticpolynucleotide in one or more microfluidic path devices held in theseating mount.

The controller be configured to perform any of the method describedherein, an in particular may be configured to receive inputs (e.g.,optical input, pressure input, temperature/thermal input, etc.) andprocess the input to control movement of fluid in the microfluidic pathdevice, temperature (including thermocycling) of various regions of themicrofluidic path device, rinsing/combining, opening/closing of valve ofthe microfluidic device, detection of the microfluidic device, etc. Thecontroller may include one or more microprocessors, communicationcircuitry, memory, etc. The controller may comprise firmware, hardwareand/or software.

Any of these apparatuses may include a one or more (e.g., a plurality)of optical sensors arranged around the seating mount and reagent storageframe to monitor fluid levels within the reagent storage frame andfluidic movement in the microfluidic path device when the microfluidicpath device is seated in the seating mount. Alternatively oradditionally, the optical sensor(s) may be present on the bottom of theapparatus (e.g., beneath the seating mount) and may be directed upwardsto detect fluid amounts, movement, etc.). The apparatus may include aseating mount release control configured to release the seating mountfrom the apparatus so that it can be separately sterilized. Any of theseapparatuses may include a fluid interface assembly release controlconfigured to release the fluid interface assembly from the apparatus sothat it can be separately sterilized, and/or a fluid sample holderrelease control configured to release the fluid sample holder from theapparatus so that it can be separately sterilized.

Any of these apparatuses may include a thermal control configured tomodulate the temperature of at least one region of the microfluidic pathdevice when the microfluidic path device is seated in the seating mount.In some apparatuses, there may be more than one thermal controlconfigured to modulate the temperature of differing regions of themicrofluidic path device. The thermal control may comprise a Peltierdevice and/or may be configured to control the temperature of the atleast one region of the microfluidic path device to between 4° C., to105° C. (e.g., between 4° C. to 99° C., between 4° C., to 98° C.,between 4° C., to 95° C., between 4° C., to 90° C., between 4° C., to85° C., between 4° C., to 80° C., between 4° C., to 75° C., between 4°C., to 70° C., between 4° C., to 65° C., between 4° C., to 60° C.,etc.).

Any of these apparatuses may include a magnetic field applicatorconfigured to apply a magnetic field to at least one region of themicrofluidic path device when the microfluidic path device is seated inthe seating mount. The magnetic field applicator may comprise a controlarm mounted to the reagent storage frame.

In general, the controller may be configured to detect an identifyingcode on the fluid vial held by the fluid sample holders; in somevariations, the identifying code comprises a barcode, RFID, or other wayto identify (particularly in a non-contact manner) the contents ofcomponents of the fluid sample holder. The controller may be configuredto determine a level of a reagent held by the reagent storage frame.

Any of these apparatuses may include an optical sensor drive configuredto move one or more of the plurality of optical sensors around theseating mount and reagent storage frame, and/or one or moreelectroluminescent panels or other backlighting devices configured toprovide illumination to a region underlying a portion of the reagentstorage frame.

The methods and apparatuses described generally include one or morefluid power circuits to move material (liquid material) between thefluid chambers (depots, fluid-contacting sides, reactors, etc.) andchannels of the microfluidic path device or within the microfluidic pathdevice. A fluid power circuit may be a hydraulic or pneumatic circuitthat may include the microfluidic device, and in particular one or morepressure channels and pressure-receiving sides of the chambers in amicrofluidic device. The fluid power circuits may also be referred to asmicrofluidic power circuits. A single microfluidic chip may includemultiple fluid power circuits; the fluid power circuits may also includeone or more pressure lines and the interface between the pressure linesof the microfluidic control apparatus and the one or more microfluidicchips within the microfluidic path device. One or more fluid powercircuits may share components (valves, pressure lines, vacuum caps,etc.) with other, overlapping fluid power circuits. Furthermore, for thesame of convenience, it should be understood that where the term“pneumatic” is used, a general fluid power circuit (e.g., hydraulicand/or pneumatic) may be used instead or additionally. The fluidmaterial being driven by the fluid power line may be any appropriatefluid (e.g., gas or liquid, such as air, water, oil, etc.).

Also described herein are microfluidic path devices for processingtherapeutic polynucleotides in a closed path (e.g., closed-pathmicrofluidic path devices). As mentioned, these microfluidic pathdevices may be referred to herein as microfluidic chips, microfluidicpath plate, process chip, biochip, process plate, etc. In general, themicrofluidic path device may be microfluidic path plate devices, whichmay be substantially flat plate-like structures; these structures may berelatively thin (e.g., less than a few mm thick, e.g., between 0.5-20 mmthick, between 0.5-15 mm thick, between 0.5-10 mm thick, etc.). Themicrofluidic path devices described herein may generally be at leastpartially transparent, and in particular, may be transparent on the topof the microfluidic path device, so that one or more optical sensors(cameras. CCD, fiber optics, etc.) may be used to sense, detect,monitor, record, etc, action, including fluid movement and/or movementof the elastic layer, with the microfluidic path device as it is used bythe microfluidic apparatuses described herein.

Any of these microfluidic path devices may be configured to operate, asdescribed herein, as closed-path devices, in which the chambers (andparticularly the fluid-contacting chambers and fluid channels are sealedto the fluid input/output lines (e.g., fluid line) by a sealingconnection that prevents exposure to the environment (e.g., air). Thismay be particularly critical in the manufacture of therapeutic mRNAswhich may be degraded by exposure to RNAses and other contaminants inthe environment.

For example a microfluidic path device may include: an elastic layersandwiched between a first surface and a second surface; a plurality ofchambers formed between the first surface and the second surface,wherein a portion of the elastic layer divides each chamber into afluid-contacting side in the second surface and a pressure-receivingside in the first surface; a plurality of fluid channels each extendingfrom a fluid port, the elastic membrane and through the first surface,and into the second surface to fluidly connect with the fluid-contactingsides of the plurality of chambers; and a plurality of pressure channelseach extending from a one or more pressure port, through the firstsurface and the elastic layer, into the second surface and back throughthe elastic layer into the first surface, wherein each pressure channelof the plurality of pressure channels fluidly connects with one or morepressure-receiving sides of the plurality of chambers, further whereinthe volumes of the fluid-contacting sides of each chamber may beadjusted by applying pressure from the one or more of the pressureports.

A microfluidic path device (e.g., for forming a therapeuticpolynucleotide, such as a therapeutic mRNA) may include: an elasticlayer sandwiched between a first plate region having a first surface anda second plate region having a second surface; a plurality of chamberseach having a fixed volume and formed between the first surface and thesecond surface, wherein a portion of the elastic layer divides eachchamber into a fluid-contacting side in the second surface and apressure-receiving side in the first surface; a plurality of fluidchannels each extending from a fluid port through the first plate regionand into the second plate region to fluidly connect with thefluid-contacting side of one or more of the plurality of chambers; and aplurality of pressure channels each extending from one or more pressureports, through the first plate region and elastic layer, into the secondplate region, and back through the elastic layer and into the firstplate region, wherein each pressure channel of the plurality of pressurechannels extends within the first plate region and fluidly connects withone or more pressure-receiving sides of one or more of the plurality ofchambers, wherein the plurality fluid-contacting sides of the pluralityof chambers are interconnected, further wherein the fluid-contactingside of each chamber is concave so that the elastic layer seats flushand without gaps to the fluid-contacting side in the second surface whena positive pressure in the pressure-receiving side drives the elasticlayer against the fluid-contacting side.

Any of these microfluidic devices may be configured to form a secureseal with one or more fluid and/or pressure lines. In some variationsthe ports (fluid ports, pressure ports, etc.) may be formed as channelsinto the body of the microfluidic device (e.g., cylindrical channels)down to an opening through the elastic layer of the device; theunderside of this elastic layer may be supported by the second plateregion (e.g., the second surface of the second plate region) with apassage into the second plate region that has a narrow diameter than theport channel diameter, so that an input line (fluid line and/or pressureline) may be supported against the elastic layer when driven against theelastic layer to form a seal.

For example, a microfluidic path device (e.g., for forming a therapeuticpolynucleotide) may generally be configured to operate in a closed path.The microfluidic path device may include: an elastic layer sandwichedbetween a first plate region having a first surface and a second plateregion having a second surface; a plurality of chambers each having afixed volume and formed between the first surface and the secondsurface, wherein a portion of the elastic layer divides each chamberinto a fluid-contacting side in the second surface and apressure-receiving side in the first surface; a plurality of fluidchannels each extending from a fluid port through the first plate regionand into the second plate region to fluidly connect with thefluid-contacting side of one or more of the plurality of chambers; and aplurality of pressure channels each extending from one or more pressureports, through the first plate region and elastic layer, into the secondplate region, and back through the elastic layer and into the firstplate region, wherein each pressure channel of the plurality of pressurechannels extends within the first plate region and fluidly connects withone or more pressure-receiving sides of one or more of the plurality ofchambers, wherein each fluid port comprises a port channel that extendsextending transversely through the first plate region, and opens onto anopening through the elastic layer having an opening diameter that issmaller than a diameter of the port channel, further wherein a diameterof the fluid channel second plate region is smaller than the diameter ofthe port channel.

Any of these device may be configured to reduce or eliminate bubblesthat may form within the fluidic pathways, e.g., by include one or morevacuum cap within the fluidic circuit(s) of the device. For example, amicrofluidic path device (e.g., for processing therapeuticpolynucleotides in a closed path) may include: an elastic layersandwiched between a first plate region having a first surface and asecond plate region having a second surface; a plurality of chamberseach having a fixed volume and formed between the first surface and thesecond surface, wherein a portion of the elastic layer divides eachchamber into a fluid-contacting side in the second surface and apressure-receiving side in the first surface; a plurality of fluidchannels each extending from a fluid port through the first plate regionand into the second plate region to fluidly connect with thefluid-contacting side of one or more of the plurality of chambers; and aplurality of pressure channels each extending from one or more pressureports, through the first plate region and elastic layer, into the secondplate region, and back through the elastic layer and into the firstplate region, wherein each pressure channel of the plurality of pressurechannels extends within the first plate region and fluidly connects withone or more pressure-receiving sides of one or more of the plurality ofchambers, at least one vacuum cap between at least some of the pluralitychambers, wherein the at least one vacuum caps comprises abubble-removing chamber formed between the first surface and the secondsurface, wherein the elastic layer divides the bubble-removing chamberinto a fluid-contacting side of the bubble-removing chamber in thesecond surface and a vacuum receiving side in the first surface, furtherwherein the fluid-contacting side of the bubble-removing chamber is influid communication with at least two of the fluid-contacting sides ofthe plurality of chambers and wherein at least the portion of theelastic layer between the fluid-contacting side of the bubble-removingchamber and the vacuum receiving side is gas permeable.

Thus, any of these microfluidic path devices may include at least onevacuum cap between at least some of the plurality chambers, wherein theat least one vacuum caps comprises a bubble-removing chamber formedbetween the first surface and the second surface, wherein agas-permeable elastic layer divides the bubble-removing chamber into afluid-contacting side of the bubble-removing chamber in the secondsurface and a vacuum receiving side in the first surface, furtherwherein the fluid-contacting side of the bubble-removing chamber is influid communication with at least two of the fluid-contacting sides ofthe plurality of chambers.

Any of these microfluidic path devices may be configured to prevent deadspace regions within even the smallest chambers of the microfluidicspath device. For example, the fluid-contacting side in the secondsurface and the pressure-receiving side are concave and configured sothat the elastic layer seats flush and without gaps to thefluid-contacting side in the second surface when a positive pressure inthe pressure-receiving side drives the elastic layer against thefluid-contacting side.

In general, these device may be formed of one plate or multiple plates.For example, a single plate may include multiple surfaces, includinginternal surfaces. Alternatively, the device may include two or moreplates that may be stacked onto each other and/or laminated together,including with an elastic layer and/or membrane between then. In somevariations of the microfluidic path device the first surface and thesecond surface may be part of at least one plate or plate region. Forexample, the first surface may be part of a first plate and the secondsurface is part of a second plate. Alternatively the first surface maybe part of a first plate region and the second surface may be part of asecond plate region; in some variations the first plate region andsecond plate region may be part of the same plate; alternatively thefirst plate region and the second plate region may be part of differentplates forming the microfluidic path device.

The one or more pressure ports and fluid ports may be disposed adjacentto a periphery of the microfluidic path device. The pressure ports andfluid ports may be arranged in groups and/or interspaced. In general,the pressure ports and fluid ports may be arranged around the peripheryof the microfluidic path device along the top of the device, and/or maybe arranged to that the central region of the microfluidic path deviceis open and exposed for visualization (by one or more optical sensors)that may monitor fluid movement and/or processing of the microfluidicpath device.

In some variations, the chambers of the microfluidic path devices may bepaired chambers, wherein a first chamber (e.g., the fluid-contactingportion) of each paired chamber of the plurality is fluidicallyconnected to a second chamber (e.g., the fluid contacting portion) ofeach paired chamber. The pressure-receiving sides of each chamber may beseparately controlled by coupling with separate (or separable and/orjoinable) pressure lines, or fluid power circuits on the microfluidicpath device. In some variations, a first chamber of a paired chamber maybe connected to any of the other paired chamber via a valved fluidicconnection. The valve may be part of a fluid power circuit and may beopened/closed by the controller applying fluid pressure (e.g.,pneumatic, hydraulic, etc.) to deflect the elastic layer within thesmall chamber formed between the first and second surfaces.

As mentioned above, the microfluidic path device may be a sealed pathdevice. The operation of the device may be monitored and controlled bythe controller apparatus without contacting the liquids (e.g.,containing the therapeutic polynucleotide, e.g., mRNA). In somevariations the microfluidic path device may be at least substantiallytranslucent to visible or ultraviolet light. For example, themicrofluidic path device is substantially transparent to visible orultraviolet light.

Any of these methods and apparatuses may be configured to purify thepolynucleotide (e.g., the mRNA) on in the microfluidic path device. Forexample, the microfluidic path device may include a material insertedinto the fluid-contacting side of the channel; e.g., the material maycomprise a cellulose material configured to selectively absorbdouble-stranded mRNA.

Any of these microfluidic path devices may be configured to removeimpurities from the therapeutic material (e.g., a “drug particle”), suchas the therapeutic mRNA material (e.g., a therapeutic mRNA encapsulatedin a delivery vehicle). For example, any of the microfluidic pathdevices described herein may include one or more chambers configured forbuffer adjustment and/or drug particle concentration. In some cases, theapparatus is configured to tangentially flow a solution of drugparticles through a chamber having one or more ultrafiltration membranesto separate nanoparticles from the solvents, thereby purifying and/orconcentrating drug particles in a retentate. Smaller particles, such assolvents and ions, can be pass through the membrane as a permeatematerial, to waste, while drug particles (e.g., mRNA encapsulated indelivery vehicle) in the same solvent can be collected downstream asretentate. In some variations this may concentrate the drug particles.In some cases, a biocompatible and stable buffer can be used fordownstream processing for injection to patients. Buffer adjustment maybe accomplished by adding diluent with appropriate composition of water,salt, excipients, and/or other constituents. The concentration ofcertain chemicals may be increased by adding a buffer with higherchemical concentration and vice versa. For example, in some cases,ethanol concentration can be reduced by half by adding the same volumeof water. The methods and apparatuses described herein can allow for theformulation of the biomolecule-containing product, buffer adjustment andconcentration to be performed in one microfluidic path device. Theformulation buffer may be adjusted to a more biocompatible and stablebuffer for downstream processing and injection to patients. The drugconcentration may be also adjusted to an acceptable volume for the drugadministration method after formulation and buffer adjustment process.Thus, any of these apparatuses may include one or more chambers having amembrane, such as an ultrafiltration membrane. A microfluidic pathdevice may therefore include a concentrator. In some variations, themicrofluidic path includes a dialysis chamber (e.g., within thethickness of the second surface, e.g., second layer portion).

Any of these microfluidic path devices may include a delivery reservoirconfigured to deliver a pre-selected volume of a fluid to the at leastone chamber; for example, a pre-selected volume of the chambers may be,e.g., between about 20 nanoliters and 5 mL (e.g., 25 nL and 5 mL, about50 nL, and 5 mL, between about 50 nL and 2 mL, greater than about 25 nL,about 30 nL, about 50 nL, about 75 nL, etc.).

The first layer portion and/or the second layer portion may be formedfrom a rigid material. Any of these microfluidic path device may includea third layer portion (e.g., a third surface) that may be formed from arigid material, e.g., laminated to an elastic material. The rigidmaterial may be a polymer, e.g., cycloolefin copolymer, or glass.

For example, a microfluidic path device may include: an elastic layerbetween (e.g., sandwiched between) a first plate and a second plate; aplurality of chambers each having a fixed volume, each chamber formedbetween the first plate and the second plate, wherein a portion of theelastic layer divides each chamber into a fluid-contacting side and apressure-receiving side; a plurality of fluid ports through the firstplate each comprising an exposed portion of the elastic layer that issupported by the second plate, wherein each fluid port comprises anopening through the elastic layer and into the second plate that fluidlyconnect with the fluid-contacting side of one of the plurality ofchambers; a plurality of pressure ports through the first plate eachcomprising an exposed portion of the elastic layer that is supported bythe second plate, wherein each fluid port comprises an opening throughthe elastic layer and into the second plate that fluidly connect withthe pressure-receiving side of one of the plurality of chambers.

A microfluidic path device may include: an elastic layer sandwichedbetween a first plate and a second plate; a plurality of chambers eachhaving a fixed volume, each chamber formed between the first plate andthe second plate, wherein a portion of the elastic layer divides eachchamber into a fluid-contacting side and a pressure-receiving side; aplurality of fluid ports each passing through the first plate andthrough the elastic layer and into the second plate to fluidly connectwith the fluid-contacting side of one of the plurality of chambers; aplurality of pressure ports each passing through the first plate andthrough the elastic layer and into the second plate, then back throughthe elastic layer and into the first layer to fluidly connect with thepressure-receiving side of one of the plurality of chambers.

For example, a microfluidic path device may include: a first platehaving a first surface and a second surface and a thicknesstherebetween; a second plate having a first surface and a second surfaceand a thickness therebetween; an elastic layer sandwiched between thesecond surface of the first plate and the first surface of the secondplate; a third plate coupled to the second plate on the second surfaceof the second plate, the third plate having a first surface and a secondsurface; at least one chamber having a fixed volume, the at least onechamber formed in the second surface of the first plate and the firstsurface of the second plate, wherein a portion of the elastic layerdivides the at least one chamber into a fluid-contacting side and apressure-receiving side; a fluid channel extending from a fluid portpassing through the thickness of the first plate, to a fluid channelopening through the elastic layer and through the thickness of thesecond plate to fluidly connect with a connecting channel formed in thesecond surface of the second plate, wherein the fluid channel connectsto the fluid-contacting side of the at least one chamber; wherein thediameter of the fluid port through the thickness of the first plate islarger than the diameter of the fluid channel opening through theelastic layer; and an exit channel extending from the fluid-contactingside through the second surface of the second plate, wherein a port(e.g., valve) formed by the elastic layer is between thefluid-contacting side and the exit channel.

For example, a microfluidic path device for processing therapeuticpolynucleotides in a closed path may include: a first plate having afirst surface and a second surface and a thickness therebetween; asecond plate having a first surface and a second surface and a thicknesstherebetween; an elastic layer sandwiched between the second surface ofthe first plate and the first surface of the second plate; a third platecoupled to the second plate on the second surface of the second plate;at least one chamber having a fixed volume, the at least one chamberformed in the second surface of the first plate and the first surface ofthe second plate, wherein a portion of the elastic layer divides the atleast one chamber into a fluid-contacting side and a pressure-receivingside; a fluid channel extending from a fluid port passing through thethickness of the first plate, to a fluid channel opening through theelastic layer and through the thickness of the second plate to fluidlyconnect with a connecting channel formed in the second surface of thesecond plate and bounded by the third plate, wherein the fluid channelconnects to the fluid-contacting side of the at least one chamber; apressure channel extending from a pressure port passing through thethickness of the first plate, to a pressure channel opening through theelastic layer and into the thickness of the second plate to fluidlyconnect with a connecting pressure channel formed in the second surfaceof the first plate and bounded by the elastic layer, wherein thepressure channel connects to the pressure-receiving side of the at leastone chamber; wherein the diameter of the fluid port passing through thethickness of the first plate is larger than the fluid channel openingthrough the elastic layer, further wherein the diameter of the pressureport passing through the thickness of the first plate is larger than thepressure channel opening through the elastic layer; and an exit channelextending from the fluid-contacting side through the second surface ofthe second plate, wherein a valve (e.g., port) formed by the elasticlayer is between the fluid-contacting side and the exit channel.

A microfluidic path device may include a pressure channel extending froma pressure port passing through the thickness of the first plate, to apressure channel opening through the elastic layer and into thethickness of the second plate to fluidly connect with a connectingpressure channel formed in the second surface of the first plate andbounded by the elastic layer, wherein the pressure channel connects tothe pressure-receiving side of the at least one chamber.

The microfluidic path devices described herein may include a pluralityof pressure ports and fluid ports are disposed adjacent to a peripheryof the microfluidic path device.

For example, a microfluidic path device for processing therapeuticpolynucleotides in a closed path may include: a first plate having afirst surface and a second surface and a thickness therebetween, thefirst surface forming an exposed outer surface; a second plate having afirst surface and a second surface and a thickness therebetween; anelastic layer sandwiched between the second surface of the first plateand the first surface of the second plate; a third plate coupled to thesecond plate on the second surface of the second plate, the third platehaving a first surface and a second surface and a thicknesstherebetween, the second surface forming an exposed bottom surface onthe bottom of the device; at least one pair of chambers, each having afixed volume, the at least one pair of chambers formed in the secondsurface of the first plate and the first surface of the second plate,wherein a portion of the elastic layer bifurcates each of the at leastone pair of chambers into a fluid-contacting side and apressure-receiving side, wherein the each least one pair of chambers isfluidically connected therebetween; a fluid channel extending from afluid port passing through the thickness of the first plate, to a fluidchannel opening through the elastic layer and through the thickness ofthe second plate to fluidly connect with a connecting channel formed inthe second surface of the second plate and bounded by the third plate,wherein the fluid channel connects to the fluid-contacting side of theat least one chamber; a pressure channel extending from a pressure portpassing through the thickness of the first plate, to a pressure channelopening through the elastic layer and into the thickness of the secondplate to fluidly connect with a connecting pressure channel formed inthe second surface of the first plate and bounded by the elastic layer,wherein the pressure channel connects to the pressure-receiving side ofthe at least one chamber; and an exit channel extending from thefluid-contacting side through the second surface of the second plate,wherein a valve (e.g., port) formed by the elastic layer is between thefluid-contacting side and the exit channel.

Also described herein are apparatuses (e.g., systems) that include bothany of the microfluidic apparatuses (e.g., microfluidic path devicecontrol systems) and one or more microfluidic path devices. For example,a system may include: a microfluidic apparatus, wherein the apparatuscomprises: a seating mount for a microfluidic path device; a fluidinterface assembly comprising a plurality of fluidic lines and pressurelines, wherein each fluidic line and each pressure line is configured tobe driven against the microfluidic path device seated in the seatingmount; a reagent storage frame comprising a plurality of fluid sampleholders each configured to hold a fluid vial and each configured tocouple to the fluid interface assembly through one of the fluidic linesof the fluid interface assembly. As described in greater detail below,in some variations, the fluid sample holders may be adapted to be drivendirectly against the microfluidic path device, even without a separatefluidic line; the sample holder may form the fluidic line. The fluidicline, or separately the sample holder (e.g., vial, container, etc.) maybe configured to be held by pressure against an elastomeric seat that isformed in the microfluidic path device, e.g., at the port.

The apparatus may also include a plurality of optical sensors arrangedaround the seating mount and reagent storage frame to monitor fluidlevels within the reagent storage frame and fluidic movement in themicrofluidic path device when the microfluidic path device is seated inthe seating mount; and a controller configured to control theapplication of pressure through the pressure lines to drive fluidicmovement in the microfluidic path device when the microfluidic pathdevice is seated in the seating mount; and the microfluidic path device,the microfluidic path device comprising: a first plate having a firstsurface and a second surface and a thickness therebetween, the firstsurface forming an exposed outer surface; a second plate having a firstsurface and a second surface and a thickness therebetween; an elasticlayer sandwiched between the second surface of the first plate and thefirst surface of the second plate; a third plate coupled to the secondplate on the second surface of the second plate, the third plate havinga first surface and a second surface and a thickness therebetween, thesecond surface forming an exposed bottom surface on the bottom of thedevice; at least one chamber having a fixed volume, the at least onechamber formed in the second surface of the first plate and the firstsurface of the second plate, wherein a portion of the elastic layerbifurcates the at least one chamber into a fluid-contacting side and apressure-receiving side; a fluid channel extending from a fluid portpassing through the thickness of the first plate, to a fluid channelopening through the elastic layer and through the thickness of thesecond plate to fluidly connect with a connecting channel formed in thesecond surface of the second plate and bounded by the third plate,wherein the fluid channel connects to the fluid-contacting side of theat least one chamber; a pressure channel extending from a pressure portpassing through the thickness of the first plate, to a pressure channelopening through the elastic layer and into the thickness of the secondplate to fluidly connect with a connecting pressure channel formed inthe second surface of the first plate and bounded by the elastic layer,wherein the pressure channel connects to the pressure-receiving side ofthe at least one chamber; wherein the diameter of the fluid port passingthrough the thickness of the first plate is larger than the fluidchannel opening through the elastic layer, further wherein the diameterof the pressure port passing through the thickness of the first plate islarger than the pressure channel opening through the elastic layer; andan exit channel extending from the fluid-contacting side through thesecond surface of the second plate, wherein a valve formed by theelastic layer is between the fluid-contacting side and the exit channel.

Also described herein are methods of using any of these apparatus anddevices. For example, a method of processing a fluid in a microfluidicpath device to form a therapeutic polynucleotide (e.g., therapeuticmRNA) may include: sealingly and independently coupling a distal end ofeach of a plurality of fluid lines and a plurality of pressure lines toplurality of fluid ports or pressure ports on a surface of amicrofluidic path device, wherein each distal end is biased to be drivenagainst an elastic layer between a first surface and a second surface,wherein the microfluidic path device comprises a plurality of chamberseach divided into a fluid-contacting side formed in the second surfaceand a pressure-receiving side formed in the first surface; and drivingfluid through the fluid-contacting sides of the plurality of chambers bythe application of positive and negative pressure within thepressure-receiving sides of the chambers to change the sizes of theplurality of fluid-contacting sides.

A method of processing a fluid in a microfluidic path device mayinclude: sealingly and independently coupling a distal end of each of aplurality of fluid lines and a plurality of pressure lines to pluralityof fluid ports or pressure ports on a surface of a microfluidic pathdevice, wherein each distal end is biased to be driven against anelastic layer between a first plate and a second plate, wherein themicrofluidic path device comprises a plurality of chambers each dividedinto a fluid-contacting side formed in the second plate and apressure-receiving side formed in the first plate, wherein thefluid-contacting sides are interconnected; and driving fluid through theinterconnected fluid-contacting sides and operating a valve to meter themovement of fluid between the fluid-contacting sides of the plurality ofchambers by the application of positive and negative pressure within thepressure-receiving sides of the chambers to change the sizes of theplurality of fluid-contacting sides.

Driving fluid through the fluid-contacting sides may include deflectingan elastic layer sandwiched between the first surface and the secondsurface.

As mentioned above, any of these methods may include optical feedbackfrom the microfluidic path device to control the application of positiveand negative pressure.

These method may include controlling valves by deflecting an elasticlayer between the first surface and the second surface. For example, thecontroller may control fluid power (e.g., pneumatic, hydraulic) via afluid power circuit (e.g., fluid line, valve(s), etc.) to controlprocessing of the microfluidic path device.

In general, driving may comprise driving fluid through interconnectedfluid-contacting sides and operating a valve to meter the movement offluid between the fluid-contacting sides of the plurality of chambers bythe application of positive and negative pressure. The fluid-contactingsides may be interconnected.

For example described herein are methods of processing a fluid in amicrofluidic path device, the method comprising: sealingly andindependently coupling a distal end of each of a plurality of fluidlines and a plurality of pressure lines to plurality of fluid ports orpressure ports on a surface of a microfluidic path device, wherein eachdistal end is biased (e.g., spring-loaded) to drive the distal endsagainst an elastic layer between a first plate and a second plate,wherein the microfluidic path device comprises a plurality of chamberseach chamber divided into a fluid-contacting side formed in the secondplate and a pressure-receiving side formed in the first plate; andpneumatically driving fluid through a plurality of fluid-contactingsides of a plurality of chambers by coordinating the application ofpositive and negative pressure within the pressure-receiving sides ofthe chambers to change the sizes of the plurality of fluid-contactingsides.

In some variations, a method of processing a fluid in a microfluidicpath device may include: sealingly (and independently) coupling a distalend of each of a plurality of fluid lines and a plurality of pressurelines to plurality of fluid ports or pressure ports on a surface of amicrofluidic path device, wherein each distal end is biased to drive thedistal ends against an elastic layer between a first plate and a secondplate of the microfluidic path device, wherein the microfluidic pathdevice comprises a plurality of chambers each chamber divided into afluid-contacting side formed in the second plate and apressure-receiving side formed in the first plate; and driving fluidthrough a plurality of fluid-contacting sides of a plurality ofchambers.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a schematic of one variation of a system including amicrofluidic apparatus and a microfluidic path device as describedherein.

FIGS. 2A-2B show one example of a front and the back of a closed pathmicrofluidic apparatus for processing therapeutic polynucleotides at apoint of care.

FIG. 2C is an example of an apparatus as described herein.

FIG. 3 is a partial exploded view of one example of a closed pathmicrofluidic apparatus for processing therapeutic polynucleotides at apoint of care, as described herein, including a reagent storage frame,fluid interface assembly, thermal control and sensor assembly, and amicrofluidic path device.

FIG. 4A is an isometric view of one example of a reagent storage frame.

FIG. 4B is a side view of the reagent storage frame of FIG. 4A.

FIG. 5A is an isometric view of one example of an upper surface (e.g.,top) of a reagent storage frame.

FIG. 5B is an isometric view of the lower surface (e.g., bottom) of thereagent storage frame of FIG. 5A.

FIG. 6A is a top view of an example of a fluid interface assembly.

FIG. 6B is a bottom view of the fluid interface assembly of FIG. 6A.

FIGS. 7A-7B are perspective and side views, respectively of a tubingcompression connector assembly configured as a bias (e.g., a springbias) that is configured to urge a fluidic line against a microfluidicpath device to make a sealing connection thereto.

FIG. 7C is a graphical representation of tubing held engaged within atubing compression connector (e.g., collet) similar to that shown inFIGS. 7A-7B.

FIGS. 7D-7E illustrate an example of a fluid cartridge that may be heldin spring contact with port (e.g., fluid port) of a microfluidic pathdevice. FIG. 7E shows an enlarged view of the port interface region.

FIG. 8A is an isometric top view of one example of a seating mount, aplurality of optical sensors arranged around the seating mount on agantry, and a thermal control beneath the seating mount, as describedherein.

FIG. 8B is a side view of the sub-assembly (including the seating mount,optical sensors, and thermal control) of FIG. 8A.

FIG. 8C is a detail view of the seating mount according of FIG. 8A.

FIG. 9A is a top view of one example of a microfluidic path device.

FIG. 9B is an isometric view of the microfluidic path device of FIG. 9A.

FIG. 9C illustrates an enlarged example of a portion of a microfluidicpath device such as that shown in FIGS. 9A-9B, including a vacuum capfor bubble removal.

FIGS. 9D and 9E illustrate one example of a vacuum cap as descriedherein.

FIG. 9F shows another example of a microfluidic path device, configuredas a high-volume device (e.g., “a 10× device”).

FIG. 10A is a side sectional view through one example of a microfluidicpath device.

FIG. 10B is an example of section through a portion of one example of amicrofluidic path device as described herein.

FIGS. 10C-10I illustrate operation of a portion of one example of amicrofluidic path device interacting with a closed path microfluidicapparatus as described herein, shown in a side sectional view. In thisexample, a fluidic line and a pressure line engage with the microfluidicpath device which may precisely control fluid movement in themicrofluidic path device.

FIGS. 10J-10K illustrate a section through another example of a portionof a microfluidic path device, including a priming valve or priming cap,as described herein.

FIG. 11 is an exploded view of layers comprising a microfluidic pathdevice according to one embodiment of the disclosure.

FIGS. 12A-12C illustrate examples of varieties of microfluidic pathdevices as described herein.

FIGS. 12D-12F illustrate examples of variations of microfluidic pathdevices similar to those shown in FIGS. 12A-12C.

FIG. 13 show a section through an example of a microfluidic path deviceillustrating a closed fluidic path.

FIG. 14 schematically illustrates some of the functions of amicrofluidic path device controller as described herein.

FIGS. 15A-15B show top and bottom views, respectively, of an example ofa microfluidic path device including a heat spreader.

FIG. 16A shows one example of a system including a microfluidicapparatus in a class 5 isolation cabinet within a class 7 space. Thesystem may be configured as a mini-factory.

FIG. 16B illustrates the microfluidic apparatus within the class 5cabinet.

DETAILED DESCRIPTION

In general, described herein are apparatuses (e.g., systems, devices,etc.) and methods for processing therapeutic polynucleotides. Inparticular, these apparatuses and methods may be closed path apparatusesand methods that are configured to minimize or eliminate manual handlingduring operation. The closed path apparatus and methods may provide anearly entirely aseptic environment, and the components may provide asterile path for processing from initial input (e.g., template) tooutput (compounded therapeutic). Material inputs (nucleotides, and anychemical components) into the apparatus may be sterile, and may be inputinto the system without requiring virtually any manual interaction.

The methods and apparatuses described herein may generate therapeuticsat very rapid cycle times at very high degree of reproducibility. Theapparatuses described herein are configured to provide, in a singleintegrated apparatus, synthesis, purification, dialysis, compounding andconcentration of one or more therapeutic composition (including, but notlimited to therapeutic polynucleotides). All or some of these processingsteps may be performed in an unbroken fluid processing pathway, whichmay be configured as one or a series of consumable microfluidic pathdevice(s) (which may also be referred to as a microfluidic path chip,microfluidic path plate, process chip, biochip, or process plate). Thismay allow for patient-specific therapeutics to be synthesized, includingcompounding, at a point of care (e.g, hospital, clinic, pharmacy, etc.).

During operation of the apparatus the fluid path may remainsubstantially unbroken, and contamination may be substantiallyeliminated by non-contact monitoring (e.g., optically monitoring),including fluid flow measurement, mixing monitoring, etc, and bymanipulating precise microfluidic amounts (metering, mixing, etc.) usingpressure applied from a deflectable membrane on an opposite side of thefluidic chambers and channels.

These apparatuses and methods may be configured for use at a point ofcare. For example, the methods and apparatuses described herein may beconfigured for manufacturing customized therapeutic compositionsincluding one or more therapeutic polynucleotide (e.g., mRNA, microRNA,DNA, etc.).

Thus, the methods and apparatuses described herein may provide scalablepolynucleotide manufacturing, production of single patient dosages,elimination of touchpoints to limit contamination, input and processtracking for meeting clinical manufacturing requirements, and use inpoint-of-care operations for therapeutics. The microfluidicinstrumentation and processes described herein can provide majoradvantages.

Apparatus

In general, the apparatuses described herein may be microfluidicapparatuses (e.g., microfluidic control apparatuses). In somevariations, these microfluidic apparatuses may include closed pathmicrofluidic apparatus for processing therapeutic polynucleotides at apoint of care. These apparatuses may be configured to operate on one ormore microfluidic path device. The microfluidic apparatus may includeone or more microfluidic path device (e.g, process chip, formulationchip, etc.) or it may be configured for use with the microfluidic pathdevice, and thus, the microfluidic apparatus may not include themicrofluidic path device. In some variation the microfluidic apparatus(either with or without a microfluidic path device) may be referred toas a system.

In general, a microfluidic apparatus as described herein may include aseating mount for a microfluidic device, a fluid interface assemblycomprising a plurality of fluidic lines and pressure lines, a reagentstorage frame comprising a plurality of fluid sample holders eachconfigured to hold a fluid vial and each configured to couple to thefluid interface assembly through one of the fluidic lines of the fluidinterface assembly, a plurality of optical sensors arranged around theseating mount and reagent storage frame to monitor fluid levels withinthe reagent storage frame and fluidic movement in the microfluidic pathdevice, and a controller configured to control the application ofpressure through the pressure lines to drive fluidic movement in themicrofluidic path device. In any of these apparatuses, each fluidic lineand each pressure line may be configured to be driven against themicrofluidic path device seated in the seating mount.

The controller may coordinate processing, including moving one or morefluid(s) onto and on the microfluidic path device, mixing one or morefluids on the microfluidic path device, adding one or more components tothe microfluidic path device, metering fluid in the microfluidic pathdevice, regulating the temperature of the microfluidic path device,applying a magnetic field (e.g., when using magnetic beads), etc. Thecontroller may include software, firmware and/or hardware. In somevariations the controller may receive input from the user and mayprovide outputs (e.g., via a monitor, touchscreen, etc.). The controllermay communicate with a remote server, e.g., to track operation of theapparatus, to re-order materials (e.g., components such as nucleotides,microfluidic path devices, etc.), and/or to download protocols, etc.

FIG. 1 shows a diagrammatic representation of one example of a systemfor processing polynucleotides, including an apparatus having a housing103 enclosing a seating mount 115 which can hold one or moremicrofluidic path devices 111, which may be single use devices. Thehousing may be a chamber, enclosure, or the like, which may include alid or opening; when closed it may be sealed. The housing may enclose athermal regulator and/or may be configured to be enclosed in athermally-regulated environment (such as a refrigeration unit, etc.).The housing may form an aseptic barrier. In some variations the housingmay form a humidified or humidity-controlled environment.

The seating mount 115 may be configured to secure the microfluidic pathdevice using one or more pins or other components configured to hold themicrofluidic path device in a fixed and predefined orientation.

In some variations, a thermal control 113 may be located adjacent to theseating mount 115, to modulate temperature to the one or moremicrofluidic path devices 111. The thermal control may include athermoelectric component (e.g. Peltier device) and/or one or more heatsinks for controlling the temperature of all or a portion of themicrofluidic path device. In some variations, more than one thermalcontrol may be included, for separately regulating the temperature ofdifferent ones of one or more regions of the microfluidic path device.The thermal control may include one or more thermal sensors (e.g.,thermocouples, etc.) that may be used for feedback control of themicrofluidic path device and/or thermal control.

In FIG. 1 , a fluidic interface assembly 109 couples the liquid reagentsand/or pressure (e.g., gas) with a microfluidic path device 111 held inthe seating mount 115, and may assist in delivery of fluidic materialsas well as positive/negative gaseous pressure, from the pressure source117, to the interior of the microfluidic path device 111. The fluidinterface assembly may optionally assists in securing the microfluidicpath device(s), as described in greater detail below. The fluidinterface assembly may be removable coupled to the apparatus (and may beremoved or a portion may be removed) for sterilization between uses.

A reagent storage frame 107 is configured to contain a plurality offluid sample holders, each of which may hold a fluid vial configured tohold a reagent (e.g., nucleotides, solvent, water, etc.) for delivery tothe microfluidic device 111 or, alternatively, a fluid vial may beconfigured to receive a product from the interior of the microfluidicpath device 111. The reagent storage frame may be referred to as areagent rack. In some variations, the reagent rack includes a pluralityof pressure lines and/or a manifold configured to divide one or morepressure sources 117 into a plurality of pressure lines that may beapplied to the microfluidic path device an may be independently orcollectively (in sub-combinations) controlled.

The fluid interface assembly may include a plurality of fluid linesand/or pressure lines and may include a biased (e.g., spring-loaded)holder or tip that individually and independently drives each fluidand/or pressure line to the microfluidic path device when it is held inthe seating mount 115. The tubing (e.g., the fluid lines and/or thepressure lines) may be part of the fluid interface assembly and or mayconnect to the fluid interface assembly. In some variation the fluidlines comprise a flexible tubing that connects between the reagentstorage frame, via a connector that couples the vial to the tubing in alocking engagement (e.g., ferrule) and the microfluidic path device. Theends of the fluid paths, in some variations the ends of the fluidlines/pressure lines, may be configured to seal against the microfluidicpath device, e.g., at a sealing port formed in the microfluidic pathdevice, as described herein. For example, the ends of the fluid linesmay cut or formed to be flat (perpendicular in side view). The vials maybe pressurized (e.g., >1 atm pressure, such as 2 atm, 3 atm, 5 atm,etc.) to via the connector which may also connect to the pressuresource. For example, the fluid vials may be pressurized to between 1-20psig (e.g., 5 psig/20 psia, 10 psig, etc.). Negative or positivepressure may be applied; for example, a vacuum (e.g., −7 psig or 7 psia)may be applied to draw fluids back into the vials (e.g., the depots) atthe end of the process. In general the fluid vials may be driven atlower pressure than the pneumatic valves, which may prevent or reduceleakage. In some variations the difference in pressure between the fluidand pneumatic valves may be between about 1 psi and about 25 psi (e.g.,about 3 psi, about 5 psi, 7 psi, 10 psi, 12 psi, 15 psi, 20 psi, etc.).

As described in greater detail below, the fluid lines (or in somevariations the fluid vials directly) and pressure lines may be drivenagainst the ports (pressure port or fluid port) formed in themicrofluidic path device to form a seal. Each pressure line and/or fluidline (or fluid vial) may be individually driven against the valve seatin the microfluidic path with a bias force that may form a seal at theport. The bias force (which may be pressure due to a spring or otherforce-applying element) may be configured to be greater than thepressure within the fluid vial (and/or fluid line) and within thepressure line to maintain the seal without leaking. For example, thedifference in pressure between the fluid vial and the bias force may begreater than about 5 psi (e.g., greater than about 2 psi, greater thanabout 3 psi, greater than about 5 psi, greater than about 7 psi, greaterthan about 10 psi, etc.), and may be referred to as the valve closingpressure. In general, this bias force (valve closing pressure) mayexceed the fluid driving pressure, e.g., by an amount that may be designdependent (e.g., 3 psi, 5 psi, 7 psi, 10 psi, etc.). The bias force maybe constant or may be adjustable. The bias force may be applied tomaintain the seal with the port on the microfluidic path assembly. Insome variations the bias force may be adjusted based on the pressurewithin the fluid line (e.g., fluid vial) or the pressure line. The biasforce for each fluid line (or fluid vial) and pressure line may beindividually adjustable.

Each vial may be coded (e.g., by an identifier that may be read by oneor more sensors, as described below). The controller may monitor thefluid level and therefore the amount of each material in the fluidinterface assembly.

The apparatus may also include a magnetic field applicator 119, whichmay be configured to create a magnetic field at a region of themicrofluidic path device 111. One or more sensors 105, which may beoptical sensors, may be part of the apparatus, and may sense one or moreof a barcode, a fluid level within a fluid vial held within the reagentstorage frame, and fluidic movement within the microfluidic path device111 when the device is mounted within the mounting seat 115.

The sensors may make measurements of the process on the device, e.g., bymeasuring an optical indicator. In some variations visual/opticalmarkers may be used to estimate yield. For example, fluorescence may beused to detect process yield or residual material by tagging withfluorophores. Alternatively or in addition, dynamic light scattering maybe used to measure particle size distributions within a portion of themicrofluidic path device (e.g., such as a mixing portion). In somevariations, the sensor measurements may be done using one or two opticalfibers to convey light (e.g., laser light) in and detect an opticalsignal coming out. An instrument package may be mounted remotely fromthe device. Such non-contact sensing may be preferred.

In any of the methods and apparatuses described herein, the sensors(e.g., video sensors) may records all activity on the microfluidic pathdevice (e.g., chip or cartridge). For example, an entire run forsynthesizing and/or processing a material (such as a therapeutic RNA)may be recorded by one or more video sensors, including a video sensorthat may visualize the microfluidic path device, e.g., from above.Processing on the microfluidics path device may be visually tracked andthis record may be retained for later quality control and/or processing.Thus, the video record of the processing may be saved, stored and/ortransmitted for subsequent review and/or analysis.

The internal portion of the apparatus, e.g., within the housing 103, maybe further configured to be sterilizable. In particular, portions of theapparatus may be removed and individually sterilized. Sterilization maybe performed, e.g., by UV irradiation, or any other method ofsterilization that may be required to limit contamination or to meetregulatory requirements. The apparatus including the housing may behoused within a High Efficiency Particulate Air (HEPA) filteredenvironment. The apparatus including the housing may be housed within atemperature controlled enclosure.

As mentioned above, the apparatus may be controlled by controller 121,including to apply pressure through the microfluidic path device 111 toat least drive fluidic movement, amongst other tasks. The controller maybe completely or partially outside of the housing. The controller may beconfigured to include user inputs/outputs. For example, the userinterface 123 of the system may permit easy operation and direction ofthe apparatus and microfluidic path device(s).

Any of the apparatuses described herein may include all or some of thecomponents shown in FIG. 1 ; not all components may be necessary. InFIG. 1 , only some of the connections between components are shown;additional (or alternative) connections may be used.

FIG. 2A shows the front side of an apparatus 200, which may have any ofthe features of the apparatus of system 100. In FIG. 2A, housing 103(e.g., cover) is shown in an open position displaying the reagentstorage frame 107, sensors 105 (configured as cameras or optical sensorsin this example), which may monitor fill levels and detect barcodes in aregion below the horizontal plane of reagent storage frame 107, andcontrol arm 219 for magnetic field applicator 119. Another sensor(camera) 105′ is configured to be positioned over the upper surface ofthe microfluidic path device (not visible in FIG. 2A). In FIG. 2A, thecontrol arm for the magnet and the upper sensor, which may eachseparately include one or more actuator that may be connected andcontrolled by the controller, not shown, may be mounted to the reagentstorage frame. The apparatus also includes a gantry on which the sidesensors (cameras) may move to visualize the reagent containers and/orother components of the apparatus. The upper sensor may be configured toview the microfluidic path device when mounted in the apparatus and thisinformation may be used in operating the apparatus, e.g., by thecontroller.

FIG. 2B shows the rear side of apparatus 200, where electrical power andpressure may be connected through access region 203. FIG. 2C illustratesanother example of a system including a microfluidic path device.

The housing 103 may be made of any suitable material such as polymers,metals, or composites. The housing may be resistant to moisture andprotects the sterility of the enclosed components during operation ofthe apparatus. The housing may be designed to be contained within arefrigerator to preserve reagents at low temperature when the reagentsare stored on the reagent storage frame for the course of a batch orcontinuous run of the apparatus 200.

In FIG. 3 , an exploded view is provided of a portion of an apparatus,including a seating mount 115, microfluidic path device 111, fluidinterface assembly 109, and reagent storage frame 107, showing how theseportions fit together and align to provide functionality to themicrofluidic path device 111 in apparatus 300, which may be like theapparatuses of FIG. 1 or 2 , and have any of the features thereof. InFIG. 3 , the reagent storage frame 107 is shown with some reagent vialsand connectors (similar to FIG. 2A). Rods 303 are shown that connect thereagent frame 107 to the fluidic interface assembly 109 and can attach(e.g., by screws) to frame 107 and assembly 109 at receiving holesadjacent to the seating mount 115. The seating mount 115 may itselfinclude a frame into which the microfluidic path device 111 may beplaced, and aligning pins (not visible in FIG. 3 ) may securely orienteach of these vertically arranged components to the microfluidic pathdevice 111.

Two or more sets of pins may be used for alignment. For example, theseating mount 115 (also referred to as a lower nest) may have two ormore short (e.g., 1.5 mm) pins that the microfluidics path device 111may be aligned to when placed in the lower nest and may protrude upwardsabove the device. There may also be two long (e.g., 6 mm) pins that arepressed into the upper nest (e.g., fluidic interface assembly) and thatprotrude downward that mate to holes (visible) in the lower nest. Thesemay act to guide the upper nest into position so the smaller (e.g., 1.5mm) pins can then find the pin and slot features, also in the fluidicinterface assembly 109 that produce the final alignment.

The seating mount 115 may be secured to a base 305 and may permit orlimit (e.g., restrict) adjustment of the horizontal arrangement of themicrofluidic path device 111. In some variations, the microfluidic pathdevice Ill may be supported in a substantially horizontal plane, whichmay be useful to minimize pressures needed to drive fluidic movementthroughout the microfluidic path device 111. In some other variations,the microfluidic path device may be supported in an orientation that iswithin about 1, 2, 3, 4, 5, 7, 9, 10, 11, 13, or about 15 degrees of ahorizontal plane. Small deviations from a horizontal orientation mayassist in removing bubbles from the fluids within the chamber(s) andlines running through the microfluidic path device 111. In yet othervariations, the microfluidic path device may be supported in asubstantially vertical orientation with respect to a base 305, or may besupported in an orientation that is within about 1, 2, 3, 4, 5, 7, 9,10, 11, 13, or about 15 degrees of a vertical plane.

The seating mount 115, fluid interface assembly 109, and/or fluid sampleholders may be formed from any suitable materials, such as for example,polymers, glass, metal or composites. The seating mount 115, fluidinterface assembly 109, and/or fluid sample holders may be configured tobe sterilized, such as by autoclaving or gamma radiation exposure. Theapparatus 30) may further include one or more of: a seating mountrelease control configured to release the fluid interface assembly fromthe apparatus, a fluid assembly release control configured to releasethe fluid interface assembly from the apparatus, and/or a fluid sampleholder release control configured to release the fluid sample holderfrom the apparatus. These release controls may be engaged so that eachof the reagent storage frame, fluid interface assembly, and/or a fluidsample holder can be released from the apparatus so that they can beseparately sterilized. These components may be released and/orreinserted separately and/or collectively.

A thermal control 113 may be disposed under the seating mount 115,adjacent to the microfluidic path device 111. The thermal control 113may be configured to control the temperature in at least one region ofthe microfluidic path device 111 to between about 4° C., to about 105°C., or any selected temperature (or range of temperatures) therebetween.The thermal control may be any suitable temperature control such as, inone non-limiting example, a Peltier device, and/or a plurality ofPeltier devices. In general the thermal control may be configured toallow for controlling the temperature independently at different thermalregions simultaneously.

Optical sensors 105 may be disposed upon the base 305 and may beoriented to sense fluid fill levels within fluidic vials disposed withinthe reagent storage frame reducing probability of disrupted process runswithin the microfluidic path device 11. The optical sensors 105 may alsosense a barcode on a fluidic vial to positively identify the identityand/or lot number of a reagent or product vial. The optical sensors maysend the information about a fill level or a barcode to the controller121, where the information may be stored or acted upon. This can assistin providing chain-of-custody data crucial to regulatory controls ofpersonalized therapeutics.

The optical sensors 105 may be moveably disposed within a rail 307(e.g., as part of a gantry) on the base 305 and may further be operablyconnected to an optical sensor drive. The optical sensor drive may beconfigured to move one or more of a plurality of optical sensors 105around seating mount 115 and/or reagent storage frame 107. The pluralityof optical sensors 105 may be moved in unison by coordinating movementwith optical drive belt (e.g., drive chain) 309.

FIG. 4A shows the upper surface of reagent storage frame 107, which maybe used in any of the apparatuses described herein. Reagent storageframe 107 may support a magnetic field applicator 119 which may furtherinclude a control arm 219 which can control positioning of magnet 414 tobe adjacent at least one region of microfluidic path device Ill when inuse. One or more fluid sample holders 416 are disposed on the reagentstorage frame 107. In some variations, a reagent storage frame 107 mayinclude a plurality of fluid sample holders 416, each of which may havea same size or may have one or more different sizes. A fluid sampleholder 416 includes a cap that may be pressurized (e.g., to about 1 PSI,2 PSI, 3 PSI, 4 PSI, 5 PSI or more) above ambient atmospheric pressure,so that fluid from within a fluid vial contained by the fluid sampleholder may be driven into the microfluidic path device 111.Alternatively, the fluid sample holder may not be pressurized at all,but may operate at ambient atmospheric pressure. In yet anothervariation, the fluid sample holder cap may permit reduced pressure to beapplied to draw fluid or accept fluid being driven from the microfluidicpath device 111. A plurality of fluid sample holders may distributeapplied pressure to a plurality of fluid vials. The applied pressure maybe distributed down to the fluid interface assembly. The fluid sampleholder cap may include, for one non-limiting example, a Luer connectionor the like, which can provide leak free connections under pressure.Fluid sample holder 416 may be connected via fluidic lines to the fluidinterface assembly 109 for delivery into the microfluidic path device111. The fluidic lines connecting the fluid sample holder (and fluidvial held therein) and the fluid interface assembly may be configured tohave a length that is the shortest length possible to prevent wastageand lag.

The connections between the fluid sample holders, fluidic lines, andmicrofluidic path device can form a sealed and closed path that isisolated when the microfluidic path device is seated in the seatingmount. The closed path provides useful protection from contaminationwhen processing therapeutic polynucleotides.

The reagent storage frame may also provide a support to which asensor/camera support arm 418 is connected. The support arm 418 supportsan overhead sensor/camera 412 which is configured to image and detectsignals from the microfluidic path device 111. The sensor/camera 412 maybe a camera configured to record fluidic motion within the microfluidicpath device 111 and/or detect a signal emitted from within one or morechamber of the microfluidic path device.

The signal detected by the sensor operating as a signal detector may bea visible, a fluorescent, a UV absorbance, or an IR absorbance signal.The signal detector is a non-contact signal detector, e.g., it does nottouch the material emitting the signal directly. In some variations, thesignal detector is configured to measure a nanoparticle sizedistribution. The signal detector may be configured to measure dynamiclight scattering (DLS).

FIG. 4B shows a side view of the reagent storage frame 107, having ahorizontal surface 421 in which the fluid vials 422 contained by fluidsample holders 416 may be disposed. The reservoir portion of the fluidvials 422 project below the horizontal surface 421, permitting theoptical sensors to visualize an identity code 425, e.g, a barcode orRFID tag of a fluid vial as well as to sense the fluid level 427 offluid within the fluid vial (e.g., meniscus). In some variations,electroluminescent panels 429 may be positioned below the horizontalsurface 421 of the reagent storage frame 107 to provide additionalillumination to aid sensing. Also visible in FIG. 4B is the magnetic armcontroller 432 of the magnetic field applicator 119.

FIG. 5A shows a perspective view of the top surface of a fluid interfaceassembly 109 of apparatus 100, 200, 300. The fluid interface assembly109 may include a plurality of fluidic lines 534 and pressure lines 536,wherein each fluidic line and each pressure line is configured toindependently be driven against a microfluidic path device 111 seated inthe seating mount 115 to make a sealing connection thereto. For fluidiclines 534, a biased spring within fitting 538 may provide the force tokeep the fluidic line engaged against the microfluidic path device 111.FIG. 5B is a perspective view of the underside of the fluidic interfaceassembly 109, showing the fluidic lines 534 exiting from the undersideof the horizontal surface 421 of the fluid interface assembly 109, to beengaged with the microfluidic path device 111. The fluid lines and thepressure lines may have a distal end for engaging with the microfluidicpath device that is substantially flat, so as to form a good seal withthe microfluidics path device.

FIG. 6A shows the top surface of a fluid interface assembly 109. Thefluid interface assembly has a central opening 602 through which themicrofluidic path device 11 l may be imaged by one or more of theplurality of optical sensors. Pressure lines may pass through 642 may belocated at the periphery of the fluid interface assembly (pressure linesnot shown) and fluidic lines 534 and pressure lines 641 are arranged forinput to the microfluidic path device 111 around the periphery of thecentral opening 602. FIG. 6B shows the bottom surface of the fluidinterface assembly which shows the sealing end exit points 643 forfluidic lines 534 and sealing end 645 of pressure line 536 are arrangedaround a periphery of the central opening 602.

FIG. 7A shows a fitting 746 that may be used to engage a fluidic line534 against the microfluidic path device 11I seated in the seatingmount, in order to make a sealing connection within the microfluidicpath device 11I. The fitting 746 includes two mechanisms to provide arobust sealing connection that minimizes leaks while providingflexibility for the fluidic line 534 secured therein, which minimizesfluidic line stress during setup and operation of the apparatus. Aspring bias 748 may be employed to urge the fluidic line against themicrofluidic path device. Additionally, a collet 749 may be employed tourge the fluidic line 534 against the microfluidic path device 111. Themicrofluidic line 534 may be terminated in a flat cut to engage cleanlyagainst the microfluidic path device 111. In some variations, both ofthese mechanisms may be used to secure the fluidic lines. In othervariations, the spring bias 748 may be used. A collet 749 may be used toenhance gripping (e.g., in one direction), so that even as the fluidand/or pressure line is driven against the microfluidic path device, thefluid and/or pressure line may be prevented from backing out of themicrofluidic path device and breaking the seal with the microfluidicpath device. In yet other variations, other suitable connectors may beused to engage the fluidic lines against the microfluidic path devicesuch as a gasket or other kind of compression seal. FIG. 7B shows a sideview of the fitting including spring bias 748 and collet 749. FIG. 7Cshows a graphical representation of the fitting 746 having a fluidicline 534 engaged. Spring bias 748 is engaged against the fitting baseand collet 749 grips fluidic line 534. The flat cut end 735 is pushedpast the collet 749 in order to engage against the microfluidic pathdevice 111.

In some variations, all or some of the fluidic lines and fluid vials mayinstead or additionally be configured as fluid cassettes that connect tothe microfluidic path device 111. Any of these fluid vials (e.g., fluiddepots) may be configured so that the fluidic depot comprises a fluidicline integrated as part of the fluidic depot. One example is shown inFIGS. 7D-7E, where fluid cassette 750 may have a pressure port 753 and aflat cut or flat molded fluidic input port 755. This fluidic input portis configured as a channel that comprises a fluidic line 755 integratedinto the rest of the fluid depot/fluid vial 750. The fluidic input port755 (shown as an integrated fluid line in FIGS. 7D and 7E) may be heldwith bias (e.g., in some variations, spring loaded) against themicrofluidic path device 111, to contact and seal against theelastomeric layer (e.g., elastic layer) within the microfluidic pathdevice 111 at the port. As mentioned above, the bias force (alsoreferred to as the valve closing pressure) may be greater than thepressure within the fluid vial/fluid line and pressure line, to preventleaking (e.g., greater than 2 psi, 5 psi, 7 psi, 10 psi, etc.) than thepressure in the fluid line/fluid vial or pressure line. For example, theport of the microfluidic path device may be configured to receive apressure of to about 5 psig (e.g., 5 psig, 7 psig, 10 psig, 12 psig, 15psig, etc.), which may be slightly higher than the pressurization for afluid vial 422 as described herein. In some variations the fluid vial(e.g., fluid cassette) 750 may not require mounting on a separatereagent storage frame but could be spring mounted upon the device 111directly or on the fluid interface assembly 109, which could reduce oreliminate tubing. The bias contact with the elastic layer of themicrofluidic path device 111 could initiate a seal opening for the fluidcassette for use. The fluid cassette 750 could facilitate barcoding andidentification thereof. Use of a plurality of fluid cassettes 750 couldsimplify the structures needed to supply reagents within themicrofluidic path device 111. This design may enable the use of anisolating sterile inner liner within the fluid cassette 750, which couldeliminate exposure of a reagent stored therein from the gas used topressurize the fluid cassette. Alternatively, other fluid cassettedesigns could achieve the same isolation from exposure to the gas, byuse of a low sliding force piston or other structural features whichcould separate the reagent held within the fluid cassette 750 from thegas used to drive the fluid into the microfluidic path device. FIG. 7Eshows an enlargement of the fluidic input port 755, which engages themicrofluidic path device directly. In any of the variations, unless thecontext makes it clear otherwise, the phrase “fluidic lines” may includeeither or both a fluid cassette (also referred to as a fluid vial)and/or tubing connected to a fluidic source (e.g., depot, such a bottle,vial, tube, etc.).

FIG. 8A is a perspective view of a bottom substrate 800 of the apparatusof FIG. 1, 2 , or 3, showing the base 305 to which seating mount 115 anda plurality of optical sensors 105 are connected. In this view, fouroptical sensors 105 may be positioned around each edge of the base, andmay be configured to move along a gantry including one or more rails 307to image along a region surrounding the seating mount and reagentstorage frame, when in place. The optical sensors 105 may be drivenalong rail 307 by optical sensor drive 851. The optical sensors may bedriven in unison by optical sensor drive belt 309. Each of the opticalsensors 105 has an angled mirror assembly 853, which permits imaging ofa field of view 855, while minimizing the space taken up by the opticalsensor 105 itself. The bottom substrate 800 in this example alsoincludes a second set of aligning pins 857, which may help to align thefluid interface assembly and the reagent storage frame with the seatingmount. FIG. 8B is a side view showing the arrangement of the base 305,optical sensors 105 (three visible in this view), optical sensor drivebelt 309, and optical sensor drive 851. The second set of aligning pins857 frame the area around the seating mount 115, and cooling fan 859 ofthe thermal control 113 is visible beneath the seating mount 115. FIG.8C shows a detail view of components arranged on the bottom substrate800. Seating mount 115 is secured by mounting springs 865 to besupported at a desired orientation and to place the opening where themicrofluidic path device will be lodged at the level of the thermalcontrol, e.g., Peltier surface (thermal control surface) 863. Seatingmount 115 may be secured to the mounting springs 865 releasably, havingrelease levers or connectors (not shown) which will permit the seatingmount 115 and fluidic interface assembly 109 (e.g., upper nest) to beremoved and sterilized between uses, for example by autoclaving.Aligning holes 861 may be present on the seating mount 115 for aligningthe microfluidic path device 111 for proper seating within the seatingmount 115. The aligning holes may be near to the outer edge of theseating mount 115. Alignment dowel pins may also be included. Thethermal control surface 863 may also include or be configured to includea vacuum chuck; vacuum grooves may be present in the upper surface sothat vacuum may be applied to suck a microfluidic path device down ingood thermal contact with the thermal control surface 863.

In general, a seating mount may be referred to as simply a ‘seat’ and isconfigured to seat one or more microfluidic path devices, either securedor unsecured within the apparatus.

The microfluidic path device Ill may, in some variations, be supportedat a substantially horizontal orientation or may be supported at anorientation within about 1, 2, 3, 4, 5, 7, 9, 10, 11, 13, or about 15degrees of a horizontal plane, to assist with control of bubbles. Fan859 is visible below the seating mount 115.

As mentioned above, the apparatus (e.g., system, apparatus or device)may include a controller. The controller may be configured to controlthe application of pressure through the pressure lines to drive fluidicmovement in the microfluidic path device when the microfluidic pathdevice is seated in the seating mount. The controller may be configuredto be in communication with the optical sensors and may sense theidentity of a code on a fluid vial or may sense the identity of a codeon the microfluidic path device. The code on the fluid vial and/or themicrofluidic device may be an optical code or may be a RFID code. Thecontroller may be configured to be in communication with the opticalsensors and may sense a level of reagent in a fluid vial. The controllermay be configured to send instructions to the optical sensor drive toposition the optical sensors selectively for sensing a code or forsensing a fluid level within a fluid vial disposed on the reagentstorage frame. The controller may be configured to control a time ofintroduction of a reagent within the microfluidic path device. Thecontroller may be configured to control a volume of the reagent to bedriven within the microfluidic path device. The controller may beconfigured to control positive pressure of a gas to at least one fluidicinput of the microfluidic path device. The controller may be configuredto sequester at least a portion of a product within a sub-region of themicrofluidic device for export. In some variations, the controller maybe configured to perform an in vitro transcription (IVT) reaction in themicrofluidic path device. The controller may also include memory, one ormore datastores.

The apparatus may include one or more processors configured to instructand/or control the apparatus. The one or more processors may alsoanalyze information from the apparatus and/or microfluidic path device.

The apparatus may include a user interface for at least one ofinputting/exporting instructions and information about the status of theapparatus, identity of reagents within the apparatus, workflow beingperformed. In some variations, the apparatus may include a graphic userinterface configured to provide input to the processor.

The apparatus may also include a remote database for storage andretrieval of data and images. The identity codes, visual log and otherinformation may be stored in any format suitable for operation of theapparatus and/or for fulfilling regulatory requirements formanufacturing and formulating personalized therapeutics.

In general, the apparatus described herein may include one or moresingle-use microfluidic path device(s), as well as reusable componentsor sub-systems; selected portions of these sub-systems may besterilized. For example, one or more of the fluid sample holders (all ora portion thereof, such as the fluid container holder(s), tubing, etc.),fluid interface assemble (all or a portion thereof), and/or the sealingmount for a microfluidic path device (all or a portion thereof, such asthe seating portion) maybe removable, sterilizable and replaceable. Theapparatus may be configured to allow these one or more regions to bereleased and removed from the apparatus. For example, the apparatus mayinclude a seating mount release control configured to release theseating mount from the apparatus so that it can be separately sterilizedand/or a fluid interface assembly release control configured to releasethe fluid interface assembly from the apparatus so that it can beseparately sterilized, and/or a fluid sample holder release controlconfigured to release the fluid sample holder from the apparatus so thatit can be separately sterilized. The release mechanism may be a lockingrelease, one or more screws, pins, hinges, etc. Any of these apparatusesmay be configured to allow portions to lift away from other regions ofthe apparatus (either automatically, manually or semi-manually,including an integrated guide or mount that supports the variousportions of the apparatus, allowing access and removal/replacement ofcertain components such as all or part of the seating mount, fluidinterface assembly and/or fluid sample holder.

Microfluidic Path Device

The apparatuses described above are configured to support and controloperations in a microfluidic path device to perform processing ofpolynucleotides. The polynucleotides may be any kind of polynucleotides,including but not limited to ribonucleic acids, deoxyribonucleic acidsand the like. The polynucleotides may include only natural nucleotideunits or may include any kind of synthetic or semi-synthetic nucleotideunits. Processing may include, but is not limited to in-vitro synthesis,purification, concentration, formulation and analysis.

An example of a microfluidic path device for synthesizing therapeuticpolynucleotides in a closed path is shown in FIGS. 9A-9B, 9F, 10A-E, and11, where FIG. 9A is a view of an upper surface 911 of the device 900,looking down through the multiple layers forming the device 900, andFIG. 9B is a perspective view of the device 900. FIGS. 10A and 10C-10Hare side views through the layers of the device 900, illustrating oneexample of an arrangement of the layers and channels, chambers and portsformed therethrough.

FIG. 10A illustrates one example of an arrangement of the layers of amicrofluidic path that form the seals, channels, valves, and chambers,including pumping chambers. In general, these apparatuses mayadvantageously be formed of a rigid or semi-rigid plates and at leastone elastic layer. The elastic layer may be a sheet of elastic materialthat is liquid-impermeable. The elastic layer maybe somewhat gaspermeable, or may be treated to be more or less gas permeable in variousregions. Although a single continuous sheet of elastic material may beused, in some variations multiple elastic materials sheets may be used,or the ‘sheet’ may be formed of sections of multiple sheets. The layersand the elastic sheet may be laminated together. In general, chambersfor holding, valving and/or pumping fluid may be formed in the plates oneither side of the elastic layer so that the elastic layer bisects thechambers into a liquid containing side and a pressure (e.g., gas)applying side. The overall volume of chamber(s) may be constant, and maybe formed into both the first (e.g., upper) plate and the second (e.g.,lower) plate, but this volume may be divided into the pressure side andthe liquid side. By applying positive or negative pressure into thepressure side, the elastic sheet may be deformed to make reduce (down tozero, closing the chamber off) the volume of the liquid containing sideor to increase the volume of the liquid containing side (to apredetermined maximum). The pressure applying side of the chamber may beconnected, e.g., via a pressure channel in the upper plate (or betweenthe upper plate and the elastic layer) to a pressure port, e.g., forapplying negative or positive pressure. The liquid containing side maybe connected via a fluid channel to a fluid port, as described herein.As will be described in greater detail herein, both the fluid port andthe pressure port may be formed by an opening into the upper plate andthe elastic layer, allowing a sealed connection that is isolated fromthe atmosphere even when there are multiple different input lines.

In FIG. 10A, the microfluidic path device 900 includes a first (e.g.,upper) surface formed on one side of a first plate 903. The first plateincludes a first (e.g., top or upper) surface 911 and a second (bottomor lower) surface 929 and a thickness between the two. The first surface911 may form an exposed outer surface. The microfluidic path device alsoincludes a second plate 905 having a first (e.g., upper or top) surface931 and a second (e.g., lower or bottom) surface 933 and a thicknesstherebetween. An elastic layer 907 is sandwiched between the secondsurface 929 of the first plate 903 and the first surface 931 of thesecond plate 905. A third plate 909 is coupled to the second plate onthe second surface 933 of the second plate, either directly orindirectly. The third plate 909 also has a first (e.g., upper or top)surface and a second (lower or bottom) surface and a thicknesstherebetwecn. The second surface of the third plate may form a bottomsurface of the microfluidic path device. Any of the plates may be formedof multiple layers, which may be laminated or otherwise connectedtogether. For example, in FIG. 10A, the third plate 909 includes anoptional second elastic layer 913 which may help couple the third plateto the second plate; the second elastic layer 913 in this example formsthe first surface 935 of the third plate 909. The layers and platesshown in FIG. 10A may not be to scale (e.g., the elastic layer 907 maybe thinner relative to the plates).

The microfluidic path device 900 shown in FIG. 10A may also include aplurality of chambers 915, 916, 918, 920 each having a fixed volume.These chambers are formed by cut-out regions (e.g., rounded/curved cuts)into the second (bottom) surface 929 of the first plate 903 and thefirst (upper) surface 931 of the second plate 905; the elastic layer 907bifurcates these chambers 915 so that each includes a liquid containingside 917 and a pressure (e.g., gas containing) side 919. Themicrofluidic path device 900 may also include multiple liquid (e.g.,fluid) channels. In FIG. 10A, a single fluid channel 921 is shownextending from a fluid port 923 passing through the thickness of thefirst plate 903, to a fluid channel opening 925 through the elasticlayer 907 and through much of the thickness of the second plate 905 downto the bottom surface 933 of the second plate where a length of theliquid channel 921 running parallel to the bottom surface of the thirdplate is formed in the bottom surface 933 of the second plate, andbounded by the upper surface of the third plate 909.

In regard to the fluid port 923, the diameter of the opening into thefirst plate 903 forming the fluid port 923, which extends through thethickness of the first plate, may be larger than the diameter of thefluid channel opening 925 which extends through the elastic layer 907and into the liquid (e.g., fluid) channel 921. The fluid channel opening925 may be centered relative to the bottom of the fluid port opening,and may be offset from the walls of the fluid port opening by at leastthe expected wall thickness of the fluid line or fluid line couplinginterface that will connect to the fluid port.

The fluid channel 921 connects to the liquid containing side 917 of afirst chamber 915. This first chamber may be configured as a valve,which has a relatively low retaining volume (fixed volume), but can befully opened or closed by the movement of the elastic layer 907.

The microfluidic path device 900 also includes a plurality of pressurechannels that may be independently controlled to apply positive and/ornegative pressure. In FIG. 10A, a single pressure port 943 is shown,connected to the fourth chamber 920, although each of the chambers 915,916, 918 may be connected to a separate pressure port and pressurechannel for independently operating and controlling the movement of theportion of the elastic layer 907 bifurcating these chambers, to valve,and/or pump each chamber independently. In some variations the pressureports may be shared between multiple chambers. In FIG. 10A the pressure(e.g., gas) port 943 is similar to the fluid (e.g., liquid) port 925,and includes an opening completely through the first plate 903, down tothe exposed elastic layer 907, to an opening through the elastic layerforming a pressure (e.g., gas) channel opening 945. The pressure channelopening 945 is continuous with a pressure (e.g., gas) channel 947 thatextends from the pressure port 943, passing through much of thethickness of the first plate 903, and in a cut-out channel along thebottom of the second plate (or alternatively into a cut-out region inthe top of the third plate) and back up through the second plate and theelastic layer 907, to a region of the pressure channel within the firstplate that connects to the pressure (e.g., gas) containing portion 919of the fourth chamber 920. As described for the similar fluid (e.g.,liquid) port, the diameter of the pressure port 943 passing through thethickness of the first plate 903 may be larger than the diameter of thepressure channel opening 945 through the elastic layer 907, and may becentered or offset by greater than the wall thickness of a pressure lineor pressure line coupling interface that will connect to the pressureport.

In the section through a microfluidic path device 900 shown in FIG. 10A,there are multiple connections to other fluid (e.g., liquid) lines,fluid ports, pressure lines and pressure ports that are not shown, asthey may be out of the plane shown. For example, in FIG. 10A the liquidcontaining side or portion 917 of the fourth chamber may be connected toadditional valves (chambers) and/or channels, including, e.g., an exitchannel extending from the liquid containing side 917. An additionalchamber (e.g, configured as a valve), no shown may be formed asdescribed above. In some variations, an exit channel may deliver fluidfrom the one or more chamber through another fluid port (not shown) to afluid receiving depot, e.g., a vial, tube, etc. This receiving depot maybe held in the reagent storage frame.

In general, this configuration of the microfluidic path device and themicrofluidic apparatus is configured so that multiple, complex steps maybe executed by the apparatus on the microfluidics path device in a fullyenclosed (sealed and protected from atmosphere) manner, withoutrequiring any manual intervention. Fluid may be metered using thefixed-volume chambers and moved, mixed, filtered, etc, by applyingpneumatic pressure to deflect regions of the elastic layer.

Returning to FIG. 9A, a microfluidic path device 900 may include atleast one pair of chambers 953, each of which may be like clamber 920,including liquid (fluid) side 917, a pressure (e.g., gas) side 919,fluidic connections, pressure connections and fluidic/pressure lines asdescribed above. Further each of the pair may be connected to each otherby a fluidic connector 955. The fluidic connector 955 may be used incoordination with positive and/or negative pressure applied to thepressure side of the chamber(s) to drive liquid in the liquid sidebetween the two chambers to mix this liquid within each of the chambers.Deflecting the elastic layer between the fixed volume of a chamberbifurcated by the elastic layer may drive any liquid within the liquidbetween the two chambers.

Any of the microfluidic path devices described herein may include one ormore connections for an electronics, including electrical sensors, onthe device. For example, in FIG. 9A, the microfluidic path device mayinclude a region 982 that is configured to include one or moreelectrical contacts for communication with one or more sensors (or otherelectronics) on the microfluidic path device. In some variations theelectronics may include electrical circuits or the like. Theelectrically active region 982 may include one or more conductors formaking electrical contact with the apparatus; the electrical contactsmay provide power, data, etc. For example, the electrically activeregion may be configured as one or more contact pads for connecting toone or more connector pins (e.g., spring-loaded or otherwise biasedconnector pins) that may be attached to or part of the upper nest (e.g.,the fluidic interface assembly).

The microfluidic path device 900 may include more than one pair ofchambers, wherein each pair of chambers may be used for differentprocesses applied to polynucleotides. For example a first pair ofchambers 953 may be used for synthesis of the polynucleotides. A secondpair of chambers 955 may be used for purification of the synthesizedpolynucleotides. Fluid from a first pair of chambers 953 may be drivento a second pair of chambers upon application of pressure to thepressure-receiving side 919 of the respective chambers and opening avalve 959 between the first pair of chambers 953 and the second pair ofchambers 955. The valve chamber 959 may be formed by the elastic layer907 within a connector channel between the two pairs of chambers.

A microfluidic path device 900 as shown in FIGS. 9A and 9B may have aplurality of pressure ports 943 and fluid ports 923. The plurality ofpressure ports and fluid ports may be disposed adjacent to a peripheryof the microfluidic path device, and are configured to be connected tothe fluid interface assembly 109 as described above.

Ports (e.g., sealing valves) may be formed from the elastic layer asdescribed above, along the length of a connecting channel 939 (eitherpressure channel or fluid channel), such as is shown in FIG. 9A, forvalve 961, which may control timing of delivery of a reagent driven fromfluidic port 923, but when placed in series with one or more similarlyconstructed valves, may also permit metering to the chambers of thedevice. For example, in FIG. 9A, three valve chambers are shown(described in greater detail below), the first of these three valves mayact as a peristaltic pump, while the middle valve may be a meteringchamber that meters small (e.g., having a metering volume of about 10nL, 20 nL, 25 nL, 50 nL, 75 nL, 100 nL, etc.). The structure of the portand channel is illustrated in FIG. 10A, described above. The size of thechannels, and particularly the size of the chambers connected to thechannels) can meter out the volume dispensed along fluidic connectingchannel 939, 921 and delivered into the chamber 953 that is connected tothe fluidic connecting channel 939, 921. In some variations, a meteredvolume may be as little as 50 nL. Metered volumes of about 100 nL, 1microliter, 5 microliters or more may be imported. A variety of valvesizes may be pre-selected for incorporation with in the microfluidicpath device 900, and reagents may be connected to appropriate meteringsizes by user choice.

Additionally, more than one valve body 961 may be included in a rowalong fluidic connecting channel 939. A series of valves 961 may act asa peristaltic pump to move fluid, including (but not limited to) viscusfluids. The ability to function as a peristaltic pump for fluidsgenerally, may have particular advantage for moving fluid that may beviscous or contain suspended particles such as purification or capturebeads.

As mentioned, a microfluidic path device 900 may also include a deliveryor export reservoir or depot 963. In FIG. 9A, a pre-selected volume maybe formed similarly to the chamber construction described above, or maycontain only a metering side, as desired. In either case, valves may beused to meter desired volumes into the reservoir 963. Valve 965 cancontrol delivery of fluid from reservoir 963. If larger volumes aredesired, the delivery may be repeated. Alternatively, if reservoir 963was pre-selected to be an export reservoir, valve 965 may open, anddeliver fluid from chamber 957, while retaining valve 967 shut, whichpermits only the measured volume of fluid to be exported to reservoir963. This fluid may then be exported to a fluid vial on the reagentstorage frame for further processing or testing. In some variations, achamber, reservoir or depot (e.g., 963) may be configured as a meteringsection of, e.g., a 1 μL pump formed by three valve structures (967,965, 967). A chamber may be configured for export of waste, for example,from a mixing chamber 957.

An advantage of the microfluidic path device 900 can be the sealed pathnature of its construction. While fluid vials, fluidic lines and themicrofluidic path device are connected, operation of the apparatus maybe performed without any exchange of materials in or out of the system,and in particularly in/out of the fluid path of the microfluidic pathdevice for processing, including synthesizing a polynucleotide andpreparing it for biological delivery (as a therapeutic, such as drug,vaccine, etc.). Thus the entire system may operate as a closed pathand/or individual microfluidic path devices may operate in the system asa closed path (protected from the atmosphere).

Some variations of the processing that may be performed within themicrofluidic path device 900 may include purification. One variation ofpurification can include incorporating a material within the fluid side917 of a chamber or channel. The material may be configured to absorbselected moieties from the fluidic mixture in a chamber or channel. Inone variation, the material may include a cellulose material, which canselectively absorb double-stranded mRNA from a mixture. The cellulosematerial may be inserted in only one chamber of a pair of chambers, suchthat upon mixing the fluid from the first chamber of the pair to thesecond chamber, double-stranded mRNA may be effectively removed from thefluidic mixture, which can then be transferred to another pair ofchambers further downstream for further processing or export.

Some variations of the microfluidic device 900 may further include aconcentrator within a chamber, which may be disposed within thethickness of the second plate and may be in fluid communication with anexit channel such as 949. The polynucleotides may be concentrated bydriving off excess fluidic medium, and the concentrated polynucleotidemixture exported out of the microfluidic path device 900 for furtherhandling or use. In some variations, the concentrator may be a dialysischamber. For example, a dialysis membrane may be present within orbetween the plates of a microfluidic path device.

The microfluidic path device 900 may be formed of materials that are atleast substantially translucent to visible and/or ultraviolet light. Bysubstantially translucent is meant that at least 90% of light istransmitted through the material compared to a translucent material. Insome variations, the microfluidic path device 900 may be formed ofmaterials that are substantially transparent to visible and/orultraviolet light. By substantially translucent is meant that at least90% of light is transmitted through the material compared to acompletely transparent material.

As mentioned above, the first plate and/or the second plate may beformed from a rigid material. The third plate may be formed from a rigidmaterial. In some variations, the third plate may be formed from a rigidmaterial laminated to an elastic material. The plates may be formed ofthe same material, or a different material(s). For example, the rigidmaterial may be a polymer or glass. The polymer or glass may bebiocompatible, e.g., does not leach any monomers or soluble smallmolecules that are toxic to living cells. The polynucleotide productsprocessed within the microfluidic path device may be administered to ananimal, so toxic contaminants are preferably reduced or eliminated bychoice of materials. Any suitable biocompatible polymer may be used,including medical grade polycarbonate-urethane, silicone polycarbonateurethane, polyether urethane, amongst others. In some variations, thepolymer may be a cycloolefin copolymer.

FIG. 9F illustrates another example of a microfluidic path device 900similar to that shown in FIG. 9A, but configured to process largervolumes. For example the device shown in FIGS. 9A-9B may be configuredto process a particular amount per cycle of the device (e.g., 500 μg perIVT cycle), while the variation shown in FIG. 9F may be configured toprocess up to 5× this amount (e.g., 5 mg per IVT cycle). Thelarger-volume microfluidic path device shown in FIG. 9F may includechambers 953, 915 that extend slightly out of the device, so that thechambers, which are still divided up by an elastic layer intofluid-contacting sides and pressure-receiving sides, may besignificantly larger. In FIG. 9F the area is approximately the samefootprint, and may therefore be retained in the same seating mount asthe device of FIGS. 9A-9B, but may hold and process significantly morefluid volume. The controller may be adapted to automatically determinethe size(s) of the microfluidic path device chambers, and/or the type ofmicrofluidic path device (e.g., template forming, IVT processing, etc.).

As mentioned above, the microfluidic path devices may be configured sothat the chambers are formed of the upper and lower surfaces of one ormore plates that extend somewhat out of the plane of the microfluidicpath device, as compared to the variation shown in FIGS. 9A-9B. Any ofthe microfluidic path devices may be referred to herein as microfluidicpath plate devices, as mentioned above. As used herein a plate may begenerally planar, but may include one or more regions that extend up andout of the device, as shown in FIG. 9F.

FIG. 10B illustrates on example of a portion of a microfluidics pathdevice, shown partially transparent and in a perspective view. In thisexample, the device includes a first plate 903 having a first surfaceand a second surface and a thickness therebetween, a second plate 905having a first surface and a second surface and a thicknesstherebetween, and an elastic layer 907 sandwiched between the secondsurface of the first plate and the first surface of the second plate. Athird plate 909 is coupled to the second plate on the second surface ofthe second plate.

The portion of the microfluidics path device shown in FIG. JOB alsoincludes sections through three chambers 915, 914 and 918, each having afixed volume. The chambers are each formed in the second surface of thefirst plate and the first surface of the second plate, and a portion ofthe elastic layer bifurcates each chamber into a fluid holding side anda pressure-applying side. A fluid channel extends from a fluid port (notvisible in FIG. 10B), and passes through the first plate to the elasticlayer 907, to a fluid channel opening through the elastic layer andthrough most of the thickness of the second plate to fluidly connectwith a connecting channel formed in the second surface of the secondplate (or between the second plate and the third plate). This connectingchannel may be bounded by the third plate. The fluid channel thenextends back up through the second plate to connect to the fluid (e.g,liquid) holding portion one of the chambers, and preferably a chamberthat is configured as a valve. In FIG. 10B, three chambers configured asvalves are connected together, so that fluid may be pumped between themand metered in small volume (e.g., 10 nL). Negative pressure (or in somevariations, zero pressure) may be applied to the pressure holding side919 of the chamber 915 to open the valve, pulling the elastic layer upand into the upper (pressure holding) side of the chamber. Positivepressure may be applied to close the valve, deflecting the elastic layerbifurcating the chamber against the lower, curving, wall of theliquid-holding side of the chamber.

A pressure channel 947 may extend from a pressure port (not visible inFIG. 10B) that is formed as a channel through the thickness of the firstplate down to the elastic layer 907. A pressure channel opening may beformed through the elastic layer and into the thickness of the secondplate to fluidly connect with a connecting pressure channel formed inthe second surface of the first plate (not visible in FIG. 10B) orbetween the second and third plate: the pressure channel may then extendback up (in a U-shaped path) through the second plate to a channelformed in the First plate (or between the first and second plate) toconnect to the pressure-holding side of one or more chambers. Thediameter of the fluid port passing through the thickness of the firstplate is typically larger than the fluid channel opening through theelastic layer, and the diameter of the pressure port passing through thethickness of the first plate may be larger than the pressure channelopening through the elastic layer. Any of the microfluidic path devicesmay also include an exit channel extending from the fluid-holding sideof a chamber or channel through the second surface of the second plateand/or through a chamber configured as a valve that can open to permitfluid within the channel or chamber to be pumped into a depot (e.g., aholder, bottle, container, vial, tube, etc.) that may be, e.g., in therack of the reagent storage frame.

In any of the microfluidic path devices described herein, the fluid maypass from the top, though the first plate, through the seal formed bythe elastic layer and through the second plate, then along the secondplate and back up into a chamber (e.g., in some cases a chamberconfigured as a valve) bifurcated by the elastic. Similarly, thepressure flow (positive or negative) may pass from the top, though thefirst plate, though a seal formed by the elastic layer and through thesecond plate, along the bottom of the second plate, then back up throughthe second plate and elastic layer then along the bottom of the firstplate to connect to a pressure-holding side of a chamber that isbifurcated by the elastic layer. In general the elastic layer maybifurcate a chamber by driving it equally or unequally; for example, theupper (pressure) chamber may be larger or smaller than the lower(liquid-holding) chamber. The application of positive or negativepressure to control the valves and/or pump or meter fluid within thechambers may be referred to herein as pneumatic or as pneumatic barrierdeflection (“pneumodeflective”).

FIGS. 10C-10H illustrate the operation of a microfluidic path device(similar to the exemplary device shown in cross-section in FIG. 10A)engaging with a microfluidics apparatus, and in particular with aplurality of fluid 1033 and/or pressure lines 1043. In FIG. 10C, themicrofluidic path device 900 is shown in cross-section; a plurality offluid 1033 and/or pressure 1043 lines (two are visible in FIG. 10B) areshown approaching the microfluidic path device, each independentlyaligned with a pair of ports, including a fluid port 923 (to which thefluid line 1033 is aligned) and a pressure port 943 (to which a pressureline 1043 is aligned). In any of the variations described herein, thepressure and fluid lines may extend from a fluid interface assembly, asdescribed above. Each pressure and fluid line may be coupled to anindependently biased (e.g., biased towards the microfluidic path device)by a compression connector, as described above and illustrated in FIGS.7A-7C. Because each fluid and pressure connection may be independentlybiased (e.g., is separately driven towards the microfluidic pathdevice), but can be deflected up/down to push and seal against theexposed elastic layer that supported on one side by the rigid secondplate, the pressure connection may form a highly tolerant seal betweenthe pressure or fluid line, as shown in FIG. 10C. Thus, the apparatus ishighly tolerant to alignment an orientation, allowing the device to beslightly displaced and/or angled relative to the fluid and/or pressurelines. The compression connector may also apply force against thesupported elastic layer to hold the seal and prevent contamination orexposure to the outside environment. A bias element (such as a spring)may push the end of the fluid line or pressure line against themicrofluidic path device. The closer the pressure line or fluid line isto the microfluidic path device, the more force is applied, however,each fluid or pressure line may be pushed back, e.g., up, at leastslightly. Finally, the apparatus may be configured to mate with themicrofluidic path device around the periphery of the microfluidic pathdevice, which may distribute the forces and make a balanced andsupported contact with the microfluidic path device all from the sameside of the device.

In FIG. 10D the fluid line 1033 and pressure line 1043 shown are drivenagainst the fluid and pressure ports of the microfluidic path devicewith a spring bias, similar to that shown in FIGS. 7A-7D, so that thedistal open ends are driven against the flat surface of the firstelastic layer 907 of the microfluidic path device, that is supportedbeneath the elastic layer by the second layer. Each pressure and fluidline may therefore be separately driven (shown by arrows), e.g., by aspring or other biasing element, against the elastic layer, forming aseal against the elastic layer 907. Thereafter, the separate pressureand fluid lines may be independently controlled to apply fluid throughthe fluid line 939, and to valve and/or meter fluid within themicrofluidic path device. For example, in FIG. 10E, a fluid 1044 may bedriven through the fluid line 1033, though the first plate 903, throughan opening in the elastic layer 907 and into a channel through thesecond plate 905, until it reaches the first chamber 915, configured asa valve 919. The first chamber 915 includes an upper, rounded portion(pressure portion) formed in the first plate and a lower, roundedportion (fluid portion) formed in the second plate and bifurcated by aportion of the elastic layer 907. The upper and lower portions of anychamber may be rounded in shape, as described here or may be cylindricalor straight walled. In this example, the valve is opened by applyingnegative pressure through a pressure line (not shown). The first chamber915 is fluidly connected to a second chamber 916 that is configured as ametering chamber. In FIG. 10E the metering chamber 916 is opened byapplying a negative pressure in the upper pressure-receiving portion ofthe bifurcated chamber. Thus, the chamber is open maximally, and thevolume is known (e.g., 50 nL, 100 nL, 150 nL, 200 nL, 250 nL, 300 nL,500 nL, etc.). An adjacent chamber 918 may also be configured as avalve, similar to 915, and may be held closed to allow the meteringchamber to fill completely. In some variations the elastic layer may bepartially permeable to air, removing bubbles and allowing completefilling by the fluid 1044.

As mentioned above, in some variations the microfluidic path apparatusincludes one or more bubble removal chambers, and/or any of the chambersof the fluid-contacting side of the chamber may be configured as abubble removal chamber, in which bubbles within the fluid of thefluid-containing side may be removed. A bubble removal chamber may bereferred to as a vacuum cap, and may generally be configured to applynegative pressure on the opposite side of the membrane while fluid isheld within the fluid-contacting side of the chamber. The membrane maybe at least partially gas-permeable, as mentioned. Any of thepressure-receiving sides of the chambers within the microfluidic pathdevices described herein may be configured with one or more projections988 into the upper (pressure-receiving side) of the chamber that preventthe elastic layer separating the pressure-receiving side of the chamberfrom the fluid-receiving side of the chamber from seating against thetop of the pressure-receiving side. In FIG. 9C, the pressure-receivingside includes the elongate projection 988 into the pressure-receivingside so that the elastic layer cannot seal against thepressure-receiving side, thereby maximizing the surface area throughwhich the vacuum applied into the pressure-receiving side may draw gas(e.g., air) through the gas-permeable elastic layer to remove bubblesfrom the liquid.

The projection may extend any appropriate depth into thepressure-receiving side. For example, this projection, which may bereferred to as a spacer, may extend to the full depth of thepressure-receiving side, or between about 0.3 times and 1 times (e.g.,between 0.4 time and 1 times, between about 0.5 times and 1 times,between about 0.6 times and 1 times, etc.) the depth of thepressure-receiving side. In some variations, more than one projectionmay be used. The projection may be cylindrical or may have multiple arms(e.g., extending from a vertex) in order to maximize the amount ofmembrane separated from the wall(s) of the pressure-receiving side, evenwhen drawing the vacuum into the pressure-receiving side.

In some variations, the chamber formed by the pressure-receiving sideand the fluid-containing side may therefore be slightly unequal involume, as the projections into the pressure-receiving side may take upsome of the volume. Thus, the elastic layer dividing the chamber may bein contact with the vacuum through a vacuum line 987, separated from theupper surface of the pressure-receiving side, as shown in FIGS. 9C, 10Band 10J-10K (described below). In operation, the vacuum cap 938, mayremove or reduce a bubble within the line by holding fluid within thefluid-contacting side of the chamber and applying a negative pressure onthe upper (pressure-receiving) side of the chamber. As mentioned, theelastic layer dividing the chamber into the fluid-contacting side andthe pressure-receiving side may be gas permeable, so that the negativepressure removes gas from the liquid (fluidic) side by drawing gas(e.g., air, nitrogen, etc.) through the membrane overlying the fluidpath. For example, the elastic layer (which may be a membrane) in thevacuum cap may be, e.g., PolyDiMethylSilicone (PDMS) elastomer film thatis sufficiently gas permeable to allow remove gas from the liquid sideof the membrane. Fluid chambers having a fixed volume (e.g., formedbetween the first plate and the second plate) as described herein may becoupled to one or more bubble removal chambers (e.g., vacuum caps,priming caps, priming valves, etc.) and/or may be configured as bubbleremoval chambers (e.g., vacuum caps, priming caps, priming valves,etc.). In some variations the portion of the elastic layer disposedbetween the first and the second surfaces forming the chamber, whichdivides the fluid-contacting side, e.g., in the second surface (and/orsecond plate) and a pressure-receiving side in the first surface (and/orfirst plate) may be only minimally (or not at all) deflected, so that itdoes not sit flush against the surface or wall of the pressure-receivingside. For example, the upper, pressure-receiving, side may be configuredto include corners and/or one or more projections so that the elasticlayer is left exposed, rather than seating flush against the upper,pressure-receiving side. However, the fluid-receiving side may still becurved (e.g., concave) so that the elastic layer is driven flush againstit, without any holdup regions, to expel all or substantially all of thefluid in the fluid-receiving side when positive pressure is applied inthe opposite pressure-receiving side.

To remove air (e.g., bubbles), the controller may hold fluid within thevacuum cap region, e.g., by blocking valves on either or both sides(entrance and exit) of the vacuum cap. e.g., by applying positivepressure to the pressure-receiving side of the valve, and may applynegative pressure to the pressure-receiving side of the vacuum cap. Theabsolute amount of negative pressure applied (e.g., the magnitude of thenegative pressure) may be the same as or different than (e.g., lessthan) that applied to deflect the membrane in other chambers, and/orwhen applying positive pressure (e.g., the same as or different than theabsolute value of the positive pressure applied to close the valve,and/or pump). Alternatively, in some variations the membrane may beconfigured to be deflected (e.g., deflected up), against the firstsurface and/or plate, e.g., to draw fluid into the enlargedfluid-contacting side of the chamber. As mentioned, the negativepressure on the pressure-receiving side of the elastic layer may be heldto allow gas (e.g., air bubbles) to be removed through the membrane. Thecontroller may receive input (e.g., from one or more optical sensors)detecting the air in the fluid-contacting side, e.g., by detecting oneor more bubbles, and may apply vacuum in the vacuum cap until the air isgone. In some variations, the controller may hold fluid in the vacuumchamber for a period sufficient to remove all or some gas (e.g., 1second or more, 5 seconds or more, 10 seconds or more, 20 seconds ormore, 30 seconds or more, 1 minute or more, 1.5 minutes or more, 2minutes or more, 5 minutes or more, between 1 second and 5 minutes,between 2 seconds and 5 minutes, between 5 seconds and 5 minutes, etc.).In FIG. 9C, the pressure may be applied through the pressure line 987 incommunication with the pressure-receiving sides of the chamber formedbetween the first and second surface (e.g., first and second plate) ofthe device. The input(s) and/or output(s) to the vacuum cap 938 may bevalved by one or more valves 992. In FIG. 9C, Fluid may exit thefluid-contacting side from a fluid line 989 at an opposite side of thevacuum cap.

The fluid-contacting side of the chamber of the pressure cap (as withthe valves and reactors described herein) may be in fluid communicationwith a fluid port that fluidly connect with the fluid-contacting side ofeach of the chambers via one or more fluid channels, which may be in thesecond surface and/or plate. The pressure-receiving side of the vacuumcap may be in fluid communication with a pressure port extending throughthe first surface/plate (e.g., and into the surface/plate) to fluidlyconnect with the pressure-receiving port or side via a pressure channelextending through the second plate and along the first plate, asdescribed herein.

FIGS. 9D-9E illustrate another variation of a vacuum cap 938′. In thisexample the vacuum cap may be configured so that the pressure receivingside 944 of the chamber has corners, as shown. An elastic layer 948separates the chamber into the pressure receiving side 944 and thefluid-contacting side 946. In FIG. 9B, the elastic layer is in theneutral position. Fluid may be driven into the fluid-contacting side,and air may be removed by drawing a vacuum (negative pressure) into thepressure receiving side 944, as shown in FIG. 9E. The pressure receivingside in this example also includes a projection 988 that prevents theelastic layer 948 from laying against the wall(s) of the pressurereceiving side, so that a larger surface area of the elastic layer mayremain exposed, as shown in FIG. 9E, permitting more removal of airthrough the elastic layer. In some variations the device may include avariation of a vacuum cap configured as a priming valve or primingchamber. In this variation, fluid may be drawn into the chamber(s) ofthe microfluidic path device from one or more fluid depos by drawing thefluid into a channel and one or more chambers and/or a priming chamberor priming valve. FIG. 10B illustrates one example of a priming valveand FIGS. 10J-10K illustrate operation of a priming chamber or primingvalve.

In FIG. 10F, the first chamber (valve 915) may be closed by applyingpositive pressure in the upper, pressure receiving portion of the firstchamber. This limits the amount of fluid within the metering chamber 916to the precise amount; this metered fluid may then be ejected intoanother chamber (e.g., a fourth chamber, configured as a mixing chamber,etc.), as shown in FIGS. 10G-10H, by opening the valve and theholding/mixing chambers (FIG. 10G), and applying positive pressure todrive fluid from the metering chamber 916 into the holding/mixingchamber 920 (FIG. 10H). The valve 918 may then be closed, as shown inFIG. 10I. The fluid in the mixing chamber may be combined withadditional fluid (e.g., from other parts of the microfluidic pathdevice, or another metered amount of the same fluidic pathwayillustrated), mixed or otherwise processed in the chamber.

Any of the microfluidic path devices described herein may bemicrofluidic path plate devices, in which the device is substantiallythin, as described above. Thus processing in/on the plate may beperformed in substantially two dimensions (2D), including purificationof any polynucleotides (e.g., mRNA). Purification of the polynucleotidesin 2D is particularly advantageous compared to prior art techniques,which may require the use of columns and may involve steps that aredifficult or impossible to perform in a closed path environment and/orin small volumes as described herein.

In addition, as illustrated in the figures (e.g., FIGS. 10A-10I), thefluid-contacting sides (and/or the pressure-receiving side) of eachchamber may be configured to so that the elastic layer seats flush andwithout gaps to the fluid-contacting side in the second surface when apositive pressure in the pressure-receiving side drives the elasticlayer against the fluid-contacting side. In some variations the fluidcontacting sides and/or the pressure-receiving sides may be concave. Theconcavity may have a somewhat shallow, oval cross-section to permit theelastic layer to readily seat flush against the wall of the fluidcontacting side (and/or pressure-receiving side). The elastic layer maypush (e.g., seat) against the wall of the chamber so that there is nodead retention portion of the chamber (e.g., of the fluid-contactingside).

In addition to valves opening and/or closing channels, the first elasticlayer may also be used to pump fluid in/out of a chamber, as illustratedabove. For example, in some variations a chamber (e.g., accessiblethrough a fluid channel in which valves on one or both sides are open)may be provided and allowed to fill with fluid from the fluid port.Negative pressure may be applied from a pressure port that is connectedto the upper half of the bisected chamber (bisected by the elasticlayer). The application of negative pressure may help prime the deviceby drawing fluid into the channel and removing air through the elasticlayer. Thus, in any of the variations described herein, the elasticlayer may be gas permeable. Once primed, fluid may be ejected out of thechamber by opening the distal valve and applying positive pressure tothe opposite side of the elastic layer to drive fluid out of thechamber.

Any of the chambers 915, 916, 918, 920 in the example shown in FIGS.10A-10I may be configured as priming chambers or priming valves(typically priming chamber are larger than priming valves; in somevariations priming chambers may be metered). The priming valve may beused to draw fluid into the microfluidic path device and remove leadingair by introducing the fluid (e.g., driven by positive pressure fromwithin the one or more fluid depots attached to the microfluidic pathdevice) and/or by applying pumping force(s) from one or more chambers(e.g., pump chambers) within the plate. In some variations fluid ismoved primarily or initially by the application of positive pressurefrom the depots.

FIGS. 10I-10K illustrate the operation of a microfluidic deviceincluding a priming valve 938″. In FIG. 10J, the microfluidic device issimilar to that shown in FIGS. 10A-10I, but with the addition of thepriming valve in fluid communication with the fluid line 1033 input(fluid port 923). Positive pressure applied in the fluid depot connectedto the fluid line may drive fluid into the microfluidic path device, butair in the line may prevent or make it difficult to drive fluid into thedevice. As shown in FIG. 10K, once the microfluidic device has scaled tothe fluid line 1033 and the pressure line 1043 (as described above), thefluid may be driven into the channel connected to the first, primingvalve 938″. A chamber or valve 915 downstream of the priming valve maybe closed by the application of positive pressure within the pressureline connected to the chamber/valve 915 while negative pressure (vacuum)is applied to the priming valve, removing the leading air ahead of theliquid in the channel. The controller may then (e.g., after eitherallowing sufficient time based on a predetermined time and/or aftersensing, e.g., optically, that air has been removed) open the closedchamber or valve 915 and allow the primed fluid to move into the device,as described for FIGS. 10B-10I, above. In some variations, particularlywhere the elastic layer is air-permeable, air may be removed in each ofthe chambers/valves, including by the use of negative pressure in thepressure-receiving valves.

Any of the apparatuses described herein may be used as described andillustrated above. For example, the methods and apparatuses describedherein may be particularly helpful for use in generating mRNAtherapeutics using in vitro transcription (IVT), as mentioned above. Forexample, the methods and apparatuses may, in a single unbroken fluidpath, which provides an RNAse-free environment, synthetize a therapeuticcomprising one or more mRNAs. These mRNAs may be customized to anindividual patient.

EXAMPLES

Any of the apparatuses described herein may be used, for example, formanufacturing therapeutics, including in particular mRNA therapeutics.For example, a system as described herein may include an integratedhardware-software system, where each batch of therapeutic material(including both drug substance and drug product) may be produced insidededicated, single-use, disposable microfluidic path devices (which maybe referred to as chips or biochips). Therapeutic production may proceedin a sterile, closed-path system, and all the production steps may beautomated to achieve a copy-exact process. This may provide a rapidturnaround of ‘personalized’ production batches whilst providing thehigh levels of reproducibility, control and quality required for therelease of therapeutic material for clinical use.

Any of the apparatuses described herein may be used with one or moremicrofluidic path devices; in some variations different microfluidicpath devices may be used sequentially or in parallel by the sameapparatus to perform different portions of the procedure. For example,in one variations in which a therapeutic mRNA is produced a firstmicrofluidic path device may be used for DNA template production as partof a Template microfluidic path device (“template biochip”). Theresulting template may be transferred in a closed-path manner by thesystem to a second microfluidic path device (e.g., transferring from thefirst microfluidic path device to a depot in the system and/or directlyinto the second microfluidic path device). In some variations, thesecond microfluidic path device may be configured to perform in vitrotranscription of the mRNA and the purification of that material togenerate the drug substance (e.g., on an “IVT biochip” or IVTmicrofluidic path device). The product(s) from this second microfluidicpath device may then be transferred (directly or via an intermediatedepot, e.g., on the reagent storage frame) to a third microfluidic pathdevice, such a formulation microfluidic path device (e.g., “formulationbiochip”). Drug product formulation may then take place on theformulation microfluidic path device.

Each microfluidic path device may include input ports (fluid ports,pressure ports, etc.), and chambers (e.g., metering valves, reactionchambers, and purification structures) that may perform each step in themanufacturing process in a continuous and closed-path manner.

As illustrated above, the microfluidic path device may be placed intothe apparatus (e.g., system), which may include any of the elementsdescribed above. For example, returning to FIG. 2C, an apparatus (e.g.,system) may include a microfluidic path device 250, a microfluidic pathdevice management system 260 or apparatus, a control panel (e.g., userinput, monitoring and analysis), and in some variations atemperature-regulated (e.g., refrigerated) environment, such as acabinet 270. The system may support all the production activities insidethe microfluidic path device(s), such as the supply of reagents, fluidcontrol, temperature control, mixing, purification and processmonitoring. Manufacturing activities on the system may be are accessedand controlled through an application software.

The microfluidic path devices and apparatuses (e.g., systems) foroperating them described herein may function as reactors for themanufacturing steps which are performed on three distinct microfluidicpath device types, as discussed above. For example, templatemicrofluidic path devices, IVT microfluidic path devices and formulationmicrofluidic path devices may be configured to include features toperform a set of unit operations in a controlled and highly reproduciblemanner. As described above, the microfluidic path devices are typicallymultilayered structures.

For example, a microfluidic path device may be composed of cyclic olefincopolymer (COC) and silicone. The COC layers may be made of TOPAS5013L-10 and the silicone layers are made of Wacker Silpuran medicalgrade silicone. The features for each layer may be generated bymachining (prototyping phase) or injection molding (production phase).Fabrication of microfluidic path devices may include: cleaning layerswith 100% isopropanol, silicon oxide sputtering, oxygen plasmaactivation, vacuum bonding, marking (e.g., barcoding and/or RFIDlabeling) of the microfluidic path device, sterilization of theassembled microfluidic path device (e.g., by UV-C or Gamma Raysterilization), and microfluidic path device storage in sterile wafermask handling boxes. Although Oxygen Plasma exposure may sterilize theindividual layers prior to assembly, later sterilization may add anadditional level of sterility assurance. The different microfluidic pathdevice types may have different designs as shown, e.g., in FIGS. 12A-12Cshow schematic examples of template (FIG. 12A). IVT (FIG. 12B) andformulation (FIG. 12C) microfluidic path devices. All of these examplemicrofluidic path devices may share a similar basic architecture andnumber of functional elements that can be used in differentconfigurations to carry out different protocols. Functional elements mayinclude input ports, metering valves, pumps, reaction chambers, mixingstructures and purification structures as described above.

FIGS. 12D-12E illustrate alternative embodiments of the microfluidicpath devices shown in FIGS. 12A-12C. FIG. 12D shows another example viewof a template microfluidic path device, configured to form a template ina closed-path system as described herein; specifically the microfluidicpath device shown in FIG. 12D may be used for forming a synthetic (e.g.,non-bacterial) template. FIG. 12E is another example of an mRNA in vitrotranscription (IVT) microfluidic path device, similar to that shown inFIG. 12B. FIG. 12F is another example of a formulation microfluidic pathdevice similar to that shown in FIG. 12C, which may be used toencapsulate the polynucleotide (e.g., mRNA) in a delivery vehicle, asdescribed herein. Any of these microfluidic path devices may besterilized, e.g., using gamma irradiation, and may be packaged sterile.The microfluidic path device may be configured to process batches ofpredetermined sizes (e.g., about 5 mg of mRNA/2-4 days, about 50 mg ofmRNA/2-4 days, about 100 mg of mRNA/2-4 days, etc.; in some variations 2g/week).

The microfluidic path devices may interface with the control systemthrough a set of spring-loaded connections for both the reagents, aswell as pneumatic lines used for managing fluid movement and valvecontrol. The reagent and gas lines may be sealed by pressure against anelastomeric layer (elastic layer) of the microfluidic path device thatcreates a completely sealed path from reagent vials into the biochip andfrom the biochip to the export vials. FIG. 13 illustrates one example ofa sealed path that may be maintained through all of the reactions insidethe microfluidic path devices, effectively preventing any contact withthe atmosphere and minimizing the risk of contamination. FIG. 13 shows asection through an example of a microfluidic path device mounted in acontrol system, in which the microfluidic path device 1301 is seated ina seating mount 1305, with a fluid interface assembly 1307 couplingfluidic and pressure lines from a reagent storage frame 1309. The closedfluid path 1315 starts from the reagent storage depots on the reagentstorage frame 1309 and passes (via fluidic lines held by the fluidinterface assembly against the microfluidic path device 1301) into themicrofluidic path device 1301 for processing. The product then isexported off of the microfluidic path device back into a depot 1313 inthe reagent storage frame.

The microfluidic path device control system (e.g., controllerhardware/software, seating mount, fluid interface assembly, reagentstorage frame, sensors, etc.) may provide a backbone for all theelectronic and hardware components. A microfluidic path device controlsystem may be aseptic and maintain a controlled environment. The systemmay also provide an interface for loading reagents and retrievingoutputs, and may hold the microfluidic path device and provide asingle-step connection to all the actuators.

A microfluidic path device control system may also monitor and controlthe operation of the device via one or more sensors, as described above.For example, a microfluidic path device control system may scan all thereagent and microfluidic path device barcodes, and may monitor fluidlevels. The microfluidic path device control system may also automateall the microfluidic path device functions. As discussed above, thesemicrofluidic path device control systems may also generate a visualrecording of all process steps and/or may provide optical qualitycontrol (QC) analysis of intermediate process outputs.

The microfluidic path device control system (which may also be referredto herein as a management system) may include the components describedabove, such as the seating mount (“nest” or “holder”) which may beconfigured such that microfluidic path devices are correctly alignedwhen in use, e.g., so that microfluidic path devices can only beinserted in a single orientation. For example, pins (e.g., two dowelpins) and/or a notch in the nest may be matched by the shape of themicrofluidic path device. The microfluidic path device management systemmay also include vial racks to hold the reagent and export vials, adownward looking camera that records all liquid and valve movements, andproduct export. Side cameras on rails may capture barcodes and detectfluid levels, and a robotic arm, e.g., with magnets, may be controlledfor head manipulation. The microfluidic path device may be held in placewith a vacuum chuck which ensures good contact with a thermal control(e.g., Peltier device) for temperature management. Once the microfluidicpath device is in place, in some variations mating with all theconnectors may be achieved in a single step by lowering the top part ofthe microfluidic path device management system through a dowel pinguided system.

A control panel, may be configured as a main interface for allelectronic devices (e.g., CPU, Ethernet RIO device controller) as wellas the valves and manifolds for pneumatic control, and pressureregulators. In some variations, the microfluidic path device controlsystem may be held in a refrigerated container or cabinet (e.g., an ISOclass 5 safety cabinet) that may provide a microbiologically safeenclosure through HEPA air filtering and air flow management and mayensure that all reagents are kept at the correct temperature through themanufacturing process. The cabinet may also be equipped with UV lampsfor sterilization of the microfluidic path device and all the internalmicrofluidic path device management system components. In somevariations, the microfluidic path device control system, may resideinside a mini environment (e.g., a 6 ft×6 ft ISO class 5 minienvironment) that may itself be in a clean room (e.g., an ISO class 7room). Operator and system interactions, including loading reagent vialsand biochip may all be performed following aseptic manner. All reagentsand consumables may enter the area double bagged and may be wiped cleanand opened in the sterile environment, to control contamination risks.

The microfluidic path device operating system described herein may beautomated by a controller. The controller may load a process protocolthat defines types of microfluidic path device and reagents to use, andmay ensure that the correct microfluidic path device type is being used.The controller may also capture the reagents and microfluidic pathdevice identifiers (e.g., barcodes) and may ensure that the reagentshave been released for use, are not expired and are loaded in thecorrect position. The controller may also execute the sequence of stepsdefined in the protocol, automating valve, pump, and blender actuators,temperature controllers, cameras, magnetic arms, and other requiredcontrollers. The controller may also create a batch log of events andprocess parameters and may record measurements from peripheral devicesand in-line measurements involving light sources and detection systems.In some variations, this log may be stored as a full digital batchrecord in the cloud.

In use, an operator may select a protocol to run, e.g., from a libraryof preset protocols, or the user may enter a new protocol (or modify anexisting protocol). From the protocol, the controller tells the operatorwhich microfluidic path device type to use, what the vial contentsshould be, and where to place the vials in the nest. The operator mayload the microfluidic path device, the required reagents and exportvials into the system. The application may confirm the presence of therequired peripherals, identifies the microfluidic path device, and scanthe identifiers (e.g., barcodes) for each reagent and product vials,ensuring that vials match the bill-of-reagents for the selectedprotocol. After confirming the starting materials and requiredequipment, the controller may execute the protocol. During execution,valves and pumps are actuated to deliver reagents, reagents are blended,temperature is controlled, and reactions occur, measurements are made,and products are pumped to destination vials. At the conclusion of theprotocol, a production batch record is created in the cloud. Thebatch-record is encrypted, and the system measurements are uploaded tothe cloud. An example of a dataflow map in shown in FIG. 14 ,illustrating some of the functions of the controller.

As used herein, the term “processing polynucleotides” may include manytypes of manipulation, including but not limited to synthesizingpolynucleotides, purifying polynucleotides, concentrating a solutioncontaining polynucleotides, formulating polynucleotides, and anycombination thereof. As used herein, the term “substantially horizontal”when used in reference to a surface means that the surface is within+/−X degrees of horizontal relative to ground (e.g., X may be, forexample, 0.1 degree, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 5degrees, 10 degrees, etc.).

Any of the microfluidic path devices described herein may include a heatspreader on the microfluidic path device or a portion of themicrofluidic path device to even out heating in this portion of thedevice. For example, FIGS. 15A-15B show another example of amicrofluidic path device 1501 (shown here as an IVT microfluidic pathdevice similar to that shown in FIGS. 12B and 12E, discussed above), butwith a large heat spreader 1507 on the bottom side. In FIG. 15B, themicrofluidic path device includes a plurality of reactors 1503, 1505(shown as fluidically linked fluid-contacting sides of chambers formedbetween two surfaces of the microfluidic path device, as describedabove. The top of the device (shown in FIG. 15A) includesperipherally-arranged fluid ports 1509 and pressure ports 1511. Themicrofluidic path device shows a plurality of fluid power circuitsbetween the pressure ports 1511, driving valves 1512, metering chambers.1513 as well as reactors 1505, 1503. All of these chambers, valves andreactors may be formed as part of a fixed volume chamber that is formedbetween a first surface and a second surface in which an elastic layerdivides each chamber into a fluid-contacting side (the reactor, meteringchamber, etc.) in the second surface and a pressure-receiving side inthe first surface (forming part of the fluid power circuit).

In the bottom view of the microfluidic path device 1501 shown in FIG.15B, the device includes a heat spreader (e.g., a copper or other highthermal conductivity material that is attached to the bottom of themicrofluidic path device). The high thermal conductivity material maybe, e.g., copper, aluminum, silver, or materials like pyrolytic graphitewith high thermal conductivity. The heat spreader 1507 may bemechanically attached to the microfluidic path device by fastenersand/or glued in place with adhesive. In some variation the microfluidicpath device heat spreader may include or may be formed of a thermallyconductive adhesive. The microfluidic path device may be thinner underthe heat spreader to improve heat transfer into the material within themicrofluidic path device. In some variations the heat spreader may alsoincrease the stiffness of the microfluidic path device.

Although FIG. 15B shows a single heat spreader 1507 on the bottom of themicrofluidic path device, more than one heat spreader may be used. Forexample, multiple heat spreaders could be used to create differenttemperature zones. The microfluidic path device may include a plasticmaterial as part of the body (e.g., plate); plastic is generally a poorthermal conductor so it may maintain lateral temperature differencesbetween different zones of the microfluidic path device. In somevariations the microfluidic path device may underlie just the reactorregions.

In some variations the thermal transfer region is attached to a flatbottom and/or may be placed in pocket(s) in the part.

The apparatuses described herein may include and/or may be used with oneor more isolation chambers. For example in some variations theapparatuses described herein may be part of a therapeutic polynucleotidemanufacturing ‘factory’ that may produce therapeutic polynucleotides,e.g., for delivery to a subject. The therapeutic polynucleotide may be,e.g., a therapeutic mRNA. FIGS. 16A-16B illustrate one example of anapparatus that may be used by itself as a factory apparatus or that maybe used as part of a parallel manufacturing unit. In FIG. 16A theapparatus(s) 1601, 1601′ may include or may be held in a class 5isolation cabinet 1603; the isolation cabinet may itself be held withina class 7 isolation space. In FIG. 16A the cabinet includes twomicrofluidic control apparatuses 1601, 1601′. The apparatuses may bepart of an assembly factory providing copy-exact GMP units that mayautomatically manufacture therapeutic polynucleotides, such astherapeutic mRNA rapidly for patient use. These apparatuses may behightly reconfigurable and allow for rapid deployment and low costproduction. In some variations they may be deployed on-demandmanufacturing “factory” units. In some variations these apparatuses maybe set up as part of a mobile unit that may be deployed to a remote sitetemporarily or for a longer time period.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

In general, any of the apparatuses and methods described herein shouldbe understood to be inclusive, but all or a sub-set of the componentsand/or steps may alternatively be exclusive, and may be expressed as“consisting of” or alternatively “consisting essentially of” the variouscomponents, steps, sub-components or sub-steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical valuesgiven herein should also be understood to include about or approximatelythat value, unless the context indicates otherwise. For example, if thevalue “10” is disclosed, then “about 10” is also disclosed. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein. It is also understood that when a value is disclosedthat “less than or equal to” the value, “greater than or equal to thevalue” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that the throughout the application,data is provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

1.-97. (canceled)
 98. A microfluidic path device comprising: (a) a firstlayer; (b) a second layer; (c) a third layer interposed between thefirst layer and the second layer, the third layer including an elasticmembrane; (d) a plurality of chambers formed between the first layer andthe second layer, a portion of the elastic membrane dividing eachchamber into a fluid-contacting side in the first layer and apressure-receiving side in the second layer; (e) a plurality of fluidchannels providing paths for communication of fluid among thefluid-contacting sides of the plurality of chambers; and (f) a pluralityof pressure ports providing paths for communication of pneumaticpressure to the pressure receiving sides of the plurality of chambers.99. The microfluidic path device of claim 98, further comprising aplurality of fluid ports configured to paths for communication of fluidthrough the plurality of fluid channels.
 100. The microfluidic pathdevice of claim 99, the plurality of fluid ports being positioned in thefirst layer.
 101. The microfluidic path device of claim 99, the thirdlayer including a plurality of fluid openings, each fluid opening beingpositioned adjacent to a respective fluid port of the plurality of fluidports, each fluid opening being configured to provide a path forcommunication of fluid from an adjacent fluid port to a correspondingfluid channel of the plurality of fluid channels.
 102. The microfluidicpath device of claim 98, the plurality of pressure ports beingpositioned in the first layer.
 103. The microfluidic path device ofclaim 98, further comprising a plurality of pressure channels providingpaths for communication of pneumatic pressure from the plurality ofpressure ports to the pressure receiving sides of the plurality ofchambers.
 104. The microfluidic path device of claim 103, the thirdlayer including a plurality of pressure openings, each pressure openingbeing positioned adjacent to a respective pressure port of the pluralityof pressure ports, each pressure opening being configured to provide apath for communication of pneumatic pressure from an adjacent pressureport to a corresponding pressure channel of the plurality of pressurechannels.
 105. The microfluidic path device of claim 98, the elasticmembrane being operable to vary a volume of each fluid-contacting sideof the plurality of chambers.
 106. The microfluidic path device of claim98, the plurality of chambers including a first chamber and a secondchamber, a first fluid channel of the plurality of fluid channelsproviding a path for communication of fluid from the fluid-contactingside of the first chamber to the fluid-contacting side of the secondchamber, the elastic membrane being operable to form a closed valve inthe first chamber to thereby prevent communication of fluid from thefirst chamber to the second chamber via the first fluid channel. 107.The microfluidic path device of claim 98, the plurality of chambersincluding a first chamber, a second chamber, and a third chamber, afirst fluid channel of the plurality of fluid channels providing a pathfor communication of fluid from the fluid-contacting side of the firstchamber to the fluid-contacting side of the second chamber, a secondfluid channel of the plurality of fluid channels providing a path forcommunication of fluid from the fluid-contacting side of the secondchamber to the fluid-contacting side of the third chamber, the elasticmembrane being operable to peristaltically drive fluid from the secondchamber to the third chamber via the second fluid channel.
 108. Themicrofluidic path device of claim 98, the plurality of chambersincluding a bubble removal chamber, the bubble removal chamber being influid communication with the fluid-contacting side of one or more otherchambers of the plurality of chambers, the bubble removal chamber beingconfigured to provide removal of air from fluid communicated from thefluid-contacting side of one or more other chambers in fluidcommunication with the bubble removal chamber.
 109. The microfluidicpath device of claim 108, further comprising a gas-permeable elasticlayer positioned at the bubble removal chamber.
 110. The microfluidicpath device of claim 109, the gas-permeable elastic layer dividing thebubble removal chamber into a fluid-contacting side and a vacuumreceiving side, the fluid-contacting side of the bubble removal chamberbeing in fluid communication with the fluid-contacting side of the oneor more other chambers of the plurality of chambers.
 111. Themicrofluidic path device of claim 98, the first layer comprising a firstplate, the second layer comprising a second plate.
 112. The microfluidicpath device of claim 98, one or both of the first layer or the secondlayer comprising a material that is at least substantially translucentto visible or ultraviolet light.
 113. The microfluidic path device ofclaim 98, one or both of the first layer or the second layer comprisinga transparent material.
 114. The microfluidic path device of claim 98,one or both of the first layer or the second layer including a rigidmaterial.
 115. A microfluidic path device comprising: (a) a first rigidlayer; (b) a second rigid layer; (c) an elastic layer interposed betweenthe first rigid layer and the second rigid layer; (d) a plurality ofchambers formed between the first surface and the second surface, aportion of the elastic layer dividing each chamber into afluid-contacting side in the first rigid layer and a pressure-receivingside in the second rigid layer; (e) a plurality of fluid channels; and(f) a bubble removal chamber, the bubble removal chamber being in fluidcommunication with the fluid-contacting side of one or more otherchambers of the plurality of chambers, the bubble removal chamber beingconfigured to provide removal of air from fluid communicated from thefluid-contacting side of one or more other chambers in fluidcommunication with the bubble removal chamber.
 116. A microfluidic pathdevice comprising: (a) a first layer; (b) a second layer; (c) a thirdlayer interposed between the first layer and the second layer, the thirdlayer including an elastic membrane; (d) a plurality of chambers formedbetween the first layer and the second layer, a portion of the elasticmembrane dividing each chamber into a fluid-contacting side in the firstlayer and a pressure-receiving side in the second layer, thefluid-contacting side of each chamber including a concave surface; (e) aplurality of fluid channels providing paths for communication of fluidamong the fluid-contacting sides of the plurality of chambers; and (f) aplurality of pressure ports providing paths for communication ofpneumatic pressure to the pressure receiving sides of the plurality ofchambers, the elastic membrane being configured to seat against theconcave surface of one or more fluid-contacting sides of the pluralityof chambers in response to pneumatic pressure communicated via one ormore pressure ports of the plurality of pressure ports.
 117. Themicrofluidic path device of claim 116, the plurality of chambersincluding a first chamber and a second chamber, the elastic membranebeing configured to prevent communication of fluid from the firstchamber to the second chamber when the elastic membrane is seatedagainst the concave surface of the fluid-contacting side of the firstchamber or the second chamber.